Human antibodies against rabies and uses thereof

ABSTRACT

Human monoclonal antibodies that specifically bind to rabies virus, antigen binding portions thereof, and methods of making and using such antibodies and antigen binding portions thereof for treating rabies virus in a subject, are provided herein.

RELATED INFORMATION

The application is a continuation of U.S. patent application Ser. No.11/890,317, filed on Aug. 2, 2007, now U.S. Pat. No. 7,727,532, whichclaims priority to PCT Application No. PCT/US2006/003644 filed on Feb.2, 2006, and U.S. Provisional Patent Application No. 60/649,512, filedon Feb. 2, 2005, the entire contents of which are hereby incorporated byreference.

The contents of any patents, patent applications, and references citedthroughout this specification are hereby incorporated by reference intheir entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Sep. 7, 2010, is namedMJI003CN.txt, and is 41,384 bytes in size.

BACKGROUND OF THE INVENTION

Rabies is an acute progressive encephalitis caused by infection with anRNA virus of the family Rhabdoviridae (genus lyssavirus). While humanrabies fatalities are rare in developed nations (there are usually fewerthan 5 deaths in the United States each year), significant numbers ofdeaths are reported in, for example, India, where 50,000 die of thedisease and more than 500,000 are treated. Even in the United States,15,000 to 40,000 people receive anti-rabies treatment each year.Typically, dogs are the major reservoirs of the disease but othermammals such as raccoon, skunk, bat, and fox are frequent reservoirs.Transmission of the virus from an animal reservoir to human usuallyoccurs by a bite or scratch that penetrates the skin. Since rabies inhumans is almost always fatal, even a suspected infection must betreated with an aggressive post-exposure treatment regimen.

The post-exposure treatment of rabies in humans consists of proper woundcare, local administration of anti-rabies serum immunoglobulininfiltrated into and around the wound, and administration of multipledoses of rabies vaccine usually over several days and weeks (for areview of prophylaxis against rabies, see, e.g., Rupprecht and Gibbonset al., N Engl J Med 351:25 (2004)). Proper wound care can lessen theamount of virus that survives to enter the patient. Infiltration of thearea with anti-rabies serum immunoglobulin can bind to the rabies virusand help clear it thereby lessening the viral load (by passiveimmunization). Administration of multiple does of rabies vaccine (activeimmunization), usually in the form of a first dose followed bysubsequent booster doses, allow for the patient to produce a vigorousactive immunity, including humoral and cellular responses. Currentsources of anti-rabies serum immunoglobulin are obtained from the bloodof vaccinated human donors. Other sources of anti-rabies serumimmunoglobulin, for example, equine or murine, are consideredunacceptable. Current sources of rabies vaccines are produced in celllines and chemically inactivated and lyophilized. While these agents,when administered in time, are highly effective, certain obstaclesremain.

For example, there are few manufacturers of these anti-rabies agents andthey remain relatively expensive, especially in the developing worldwhere they are most needed. In addition, human anti-rabies serumimmunoglobulin, because it is harvested from the serum of human donors,must be highly purified to prevent the transmission of any adventitiousagents. Moreover, the anti-rabies vaccine requires labor intensive cellculture and extensive inactivation and purification steps. Accordingly,improved immunotherapies for treating and preventing rabies infectionare needed.

SUMMARY OF THE INVENTION

The present invention solves the foregoing problems by providing arecombinant fully human anti-rabies monoclonal antibody thatspecifically binds a broad variety of rabies virus isolates and inhibitsthe ability of the virus to infect cells.

In one embodiment, this is demonstrated by the antibodies ability toneutralize (i.e., inhibit or block) rabies virus in vitro (e.g., in aRFFIT assay). In another embodiment, this is demonstrated by theantibodies ability to inhibit rabies virus infectivity in vivo in asubject, such as an animal or a human.

Human monoclonal antibodies of the invention can be made efficiently, invirtually unlimited amounts, in highly purified form. Accordingly, theantibodies are suitable for prognosing, diagnosing, and/or treating anindividual exposed or suspected of having been exposed to rabies. Theantibodies of the invention are particularly advantageous for rabiespost exposure prophylaxis (PEP) as they eliminate the need for a donorsource of human anti-rabies serum immunoglobulin. The antibodies can beproduced using a variety of techniques for making human antibodies knownin the art. For example, as exemplified herein, the antibodies can begenerated in transgenic animals expressing human immunoglobulin genesegments, e.g., transgenic mice comprising a human Ig locus. Moreover,the antibodies can be administered alone or in combination, e.g., withan anti-rabies virus vaccine or other antibodies, to increase survivalrates of subjects (e.g., animals and humans) infected with rabies virus.

Accordingly, the invention provides several advantages that include, butare not limited to, the following:

-   -   a fully human recombinant anti-rabies antibody for prognosing,        diagnosing, and/or treating rabies virus or conducting rabies        virus post exposure prophylaxis (PEP) in a subject, e.g.,        protect from or inhibit rabies virus-mediated morbidity or        mortality in a subject;    -   a composition (e.g., pharmaceutical) and/or a kit comprising one        or more fully human recombinant anti-rabies antibodies that can        be used alone or in combination with commercially available        vaccines to treat rabies infection and/or to conduct PEP in a        subject; and    -   an improved method of passive immunotherapy for treating a        subject infected with rabies virus (e.g., in need of rabies        virus post exposure prophylaxis (PEP)) which can be used alone        or in combination with active immunotherapy (rabies vaccine).

In one embodiment, the human monoclonal antibodies or antigen bindingportions thereof of the invention specifically bind to rabies virus Gglycoprotein. Particular antibodies or antigen binding portions thereofspecifically bind to an epitope within the N-terminal half of rabiesvirus G glycoprotein. Other particular antibodies or antigen bindingportions thereof specifically bind to an epitope within the C-terminaldomain of rabies virus. Such epitopes can reside, for example, withinamino acids 1-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350,350-400, 400-450, 450-500, 500-524 of rabies virus G glycoprotein, orany interval, portion or range thereof. In one embodiment, theantibodies or antigen binding portions thereof specifically bind to anepitope within the N-terminal half of rabies virus G glycoprotein, i.e.,between about amino acid residues 19-422. In another embodiment, theepitope of the rabies G glycoprotein comprises amino acid residues336-442. In one embodiment, the rabies G glycoprotein comprises aminoacid residue 336 as well as alterations thereof, such as substitutionsor deletions.

In a related embodiment, the rabies G glycoprotein epitope comprises alinear epitope, conformational epitope, discontinuous epitope, orcombinations of such epitopes.

In another related embodiment, the rabies G glycoprotein epitopeconsists of antigenic site I, antigenic site II, antigenic site III,antigenic site minor A, or combinations of such antigenic sites, forexample, antigenic site III and minor site A.

In other embodiments, the human monoclonal antibodies or antigen bindingportions thereof can be characterized as specifically binding to rabiesvirus with a K_(D) of less than about 10×10⁻⁶ M. In a particularembodiment, the antibody or antigen binding portion thereof specificallybinds to rabies virus (e.g., a rabies virus G glycoprotein) with a K_(D)of at least about 10×10⁻⁷ M, at least about 10×10⁻⁸ M, at least about10×10⁻⁹ M, at least about 10×10⁻¹⁰ M, at least about 10×10⁻¹¹ M, or atleast about 10×10⁻¹² M or a K_(D) even more favorable.

In various other embodiments, the antibodies or antigen binding portionsthereof include a variable heavy chain region comprising an amino acidsequence at least 80%, 85%, 90%, 95%, 98%, 99% or more identical to avariable heavy chain region amino acid sequence of the antibody producedby clone 17C7 (SEQ ID NO: 1), 6G11 (SEQ ID NO: 15), 5G5, 2B10, or 1E5.

In other embodiments, the antibodies or antigen binding portions thereofinclude a variable light chain region comprising an amino acid sequenceat least 80%, 85%, 90%, 95%, 98%, 99% or more identical to a variablelight chain region amino acid sequence of the antibody produced by clone17C7 (SEQ ID NO: 2), 6G11 (SEQ ID NO: 16), 5G5, 2B10, or 1E5.

In still other embodiments, the antibodies or antigen binding portionsthereof include both a variable heavy chain region comprising an aminoacid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or more identical toa variable heavy chain region amino acid sequence of the antibodyproduced by clone 17C7 (SEQ ID NO: 1), 6G11 (SEQ ID NO: 15), 5G5, 2B10,or 1E5), and a variable light chain region comprising an amino acidsequence at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to avariable light chain amino acid sequence of clone 17C7 (SEQ ID NO: 2),6G11 (SEQ ID NO: 16), 5G5, 2B10, or 1E5.

In certain other embodiments, the antibodies or antigen binding portionsthereof specifically bind to an epitope that overlaps with an epitopebound by an antibody produced by clone 17C7, 6G11, 5G5, 2B10, or 1E5and/or competes for binding to a rabies virus, or portion thereof withan antibody produced by clone 17C7, 6G11, 5G5, 2B10, or 1E5.

The variable heavy and light chain regions of the antibodies or antigenbinding portions thereof typically include one or more complementaritydetermining regions (CDRs). These include the CDR1, CDR2, and CDR3regions. In particular embodiments, the variable heavy chain CDRs are atleast 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of theantibody produced by clone 17C7 (SEQ ID NOs: 3, 4, 5), 6G11 (SEQ ID NOs:17, 18, 19), 5G5, 2B10, or 1E5 (also shown in Table 1). In otherparticular embodiments, variable light chain CDRs are at least 80%, 85%,90%, 95%, or 99%, or more identical to a CDR of a variable light chainregion of the antibody produced by clone 17C7 (SEQ ID NOs: 6, 7, 8),6G11 (SEQ ID NOs: 20, 21, 22), 5G5, 2B10, or 1E5 (also shown in Table2).

Accordingly, particular antibodies or fragments of the inventioncomprise a variable heavy chain region that includes one or morecomplementarity determining regions (CDRs) that are at least 80%, 85%,90%, 95%, or 99%, or more identical to a CDR of a variable heavy chainregion of the antibody produced by clone 17C7 (SEQ ID NOs: 3, 4, 5),6G11 (SEQ ID NOs: 17, 18, 19), 5G5, 2B10, or 1E5 and a variable lightchain region that includes one or more CDRs that are at least 80%, 85%,90%, 95%, 99%, or more identical to a CDR of a variable light chainregion of the antibody produced by clone 17C7 (SEQ ID NOs: 6, 7, 8),6G11 (SEQ ID NOs: 20, 21, 22), 5G5, 2B10, or 1E5

The variable heavy chain region of the antibodies or antigen bindingportions thereof can also include all three CDRs that are at least 80%,85%, 90%, 95%, or 99%, or more identical to the CDRs of the variableheavy chain region of the antibody produced by clone 17C7 (SEQ ID NOs:3, 4, 5), 6G11 (SEQ ID NOs: 17, 18, 19), 5G5, 2B10, or 1E5 and/or allthree CDRs that are at least 80%, 85%, 90%, 95%, 99%, or more identicalto the CDRs of the variable light chain region of the antibody producedby clone 17C7 (SEQ ID NOs: 6, 7, 8), 6G11 (SEQ ID NOs: 20, 21, 22), 5G5,2B10, or 1E5.

In another embodiment of the invention, the human antibodies or antigenbinding portions thereof (a) include a heavy chain variable region thatis encoded by or derived from (i.e., is the product of) a human VH3-30-3 or VH 3-33 gene; and/or (b) include a light chain variable regionthat is encoded by or derived from a human Vκ gene selected from thegroup consisting of VκL6, VκL11, VκL13, VκL15, or VκL19.

Human monoclonal antibodies of the present invention include full-lengthantibodies, for example, that include an effector domain, (e.g., an Fcdomain), as well as antibody portions or fragments, such as single-chainantibodies and Fab fragments. The antibodies can also be linked to avariety of therapeutic agents (e.g., antiviral agents or toxins) and/ora label.

In another aspect, the invention features isolated polypeptides thatinclude an antigen binding portion of an antibody produced by hybridomaclone 17C7, 6G11, 5G5, 2B10, or 1E5 (also referred to herein as “17C7”,“6G11”, “5G5”, “2B10”, and “1E5”).

In another aspect, the invention features isolated nucleic acidsincluding a sequence encoding a antibody heavy chain variable regionwhich is at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQID NO: 13 or 23. The invention also features isolated nucleic acids thatinclude a sequence encoding an antibody light chain variable regionwhich is at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQID NO: 14 or 24. The invention also features expression vectorsincluding any of the foregoing nucleic acids either alone or incombination (e.g., expressed from one or more vectors), as well as hostcells comprising such expression vectors.

Suitable host cells for expressing antibodies of the invention include avariety of eukaryotic cells, e.g., yeast cells, mammalian cells, e.g.,Chinese hamster ovary (CHO) cells, NS0 cells, myeloma cells, or plantcells.

In another aspect, the invention features compositions and kits thatinclude one or more isolated human monoclonal antibodies or antigenbinding portions thereof as described herein that specifically bind torabies virus and inhibit the ability of the virus to infect mammaliancells. The composition or kit can further include one or more antibodies(e.g., human monoclonal or polyclonal antibodies) or antigen-bindingportions thereof that specifically bind to rabies virus. In oneembodiment, the polyclonal antibody or antigen binding portion thereofspecifically binds to rabies virus G glycoprotein. In a particularembodiment, the composition or kit includes both (a) an isolated humanmonoclonal antibody that specifically binds to a first rabies virusisolate; and (b) an isolated human monoclonal antibody that specificallybinds to a second rabies virus isolate.

The invention also features methods of treating rabies virus disease ina subject by administering to the subject an isolated human monoclonalantibody or antigen binding portion thereof as described herein (i.e.,that specifically binds to rabies virus) in an amount effective toinhibit rabies virus disease, e.g., rabies virus-mediated symptoms ormorbidity.

Human monoclonal antibodies or portions thereof (and compositionscomprising the antibodies or portions thereof) of the invention can beadministered in a variety of suitable fashions, e.g., intravenously(IV), subcutaneously (SC), and preferably, intramuscularly (IM) to thesubject. The antibody or antigen-binding portion thereof can beadministered alone or in combination with another therapeutic agent,e.g., a second human monoclonal antibody or antigen binding portionthereof. In one example, the second human monoclonal antibody or antigenbinding portion thereof specifically binds to a second rabies virusisolate that differs from the isolate bound to the first antibody. Inanother example, the antibody is administered together with anotheragent, for example, an antiviral agent. In another example, the antibodyis administered together with a polyclonal gamma-globulin (e.g., humangamma-globulin). In another example, the antibody is administeredbefore, after, or contemporaneously with a rabies virus vaccine.

In another aspect, the invention features methods for making an antibodyor antigen binding portion thereof that specifically binds to a rabiesvirus. In one embodiment, the method involves immunizing a transgenicnon-human animal having a genome comprising a human heavy chaintransgene and a human light chain transgene with a composition thatincludes a rabies virus, e.g., live or inactivated virus and isolatingan antibody, antibody producing cell, or antibody encoding nucleic acidfrom the animal. The rabies virus can be inactivated, for example, bychemical treatment and/or lyophilization. The method can further includeevaluating binding of the antibody to the rabies virus or rabies virus Gglycoprotein.

The invention also features methods for making the antibodies or antigenbinding portions thereof by expressing nucleic acids encoding humanantibodies in a host cell (e.g., nucleic acids encoding the antigenbinding region portion of an antibody). In yet another aspect, theinvention features a hybridoma or transfectoma including theaforementioned nucleic acids.

The invention also features a method for making a hybridoma thatexpresses an antibody that specifically binds to a rabies virus byimmunizing a transgenic non-human animal having a genome that includes ahuman heavy chain transgene and a human light chain transgene, with acomposition that includes the rabies virus or rabies virus Gglycoprotein; isolating splenocytes from the animal; generatinghybridomas from the splenocytes; and selecting a hybridoma that producesan antibody that specifically binds to rabies virus or rabies virusprotein thereof.

Treatment of humans with human monoclonal antibodies offers severaladvantages. For example, the antibodies are likely to be lessimmunogenic in humans than non-human antibodies. The therapy is alsorapid because rabies virus inactivation can occur as soon as theantibody reaches sites of infection and directly neutralizes thedisease-causing rabies virus. Human antibodies also localize toappropriate sites in humans more efficiently than non-human antibodies.Furthermore, the treatment is specific for rabies virus, and isrecombinant and highly purified and, unlike traditional therapies,avoids the potential of being contaminated with adventitious agents.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of the heavy and light chainvariable region of a recombinant, anti-rabies human antibody (i.e.,clone 17C7). These sequences correspond to SEQ ID NOs: 1 and 2,respectively. The complementarity determining regions (CDRs) for eachchain are indicated, corresponding to SEQ ID NOs: 3, 4, and 5 (of theheavy chain) and 6, 7, and 8 (of the light chain).

FIG. 2 shows the amino acid sequence of the heavy and light chainvariable region of a recombinant, anti-rabies human antibody (i.e.,clone 6G11). These sequences correspond to SEQ ID NOs: 15 and 16,respectively. The complementarity determining regions (CDRs) for eachchain are indicated, corresponding to SEQ ID NOs: 17, 18, and 19 (of theheavy chain) and 20, 21, and 22 (of the light chain).

FIG. 3 is a schematic representation of the rabies virus G recombinantglycoprotein indicating fragments that were analyzed for epitope mappingstudies. Human antibody 17C7 was determined to bind epitope(s) withinamino acid residues 19-422 as determined by immunoprecipitation andimmunoblot.

FIG. 4 shows HuMab 17C7 neutralizes rabies virus as determined by RFFITwhen diluted serially from 1:5 to 1:390625 as compared to human serum(hRIG).

FIG. 5 ERA-N and ERA-CO glycoproteins were expressed in 293T cells andreadily expressed when codon optimized (A) and capable of being bound by17C7 (B-D) when expressed on the surface of cells.

FIG. 6 shows that HuMab 17C7 recognizes the rabies G ectodomain (A) andunder non-reducing conditions (B) as well as the G protein of strain ERA(C).

FIG. 7 shows that HuMab 17C7 recognizes N336K and N336D mutant ERAglycoproteins (A) by ELISA and by immunoblot (B).

FIG. 8 shows that HuMab 17C7 neutralizes ERA pseudovirus infection ofcells (A-B) and the consequences of various mutations to the ERA Gprotein (C-D) regarding 17C7 binding thereto.

FIG. 9 shows the consequences of various mutations to the ERA G protein(A-B) regarding 17C7 binding thereto.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear understanding of the specification andclaims, the following definitions are conveniently provided below.

Definitions

As used herein, the term “rabies virus” refers to the virion or portionthereof, for example protein portion, such as rabies virus Gglycoprotein that is encoded by the RNA of rabies virus.

The term “anti-rabies virus antibody” is an antibody that interacts with(e.g., binds to) a rabies virus or a protein, carbohydrate, lipid, orother component produced by or associated with rabies virus. A “rabiesvirus G glycoprotein antibody” is an antibody that binds a Gglycoprotein of rabies virus or a fragment thereof. An anti-rabies virusor G glycoprotein antibody may bind to an epitope, e.g., aconformational or a linear epitope, or to a portion or fragment of thevirus or component thereof.

The term “human antibody” is an antibody that has variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies described herein may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo).

An anti-rabies virus antibody, or antigen-binding portion thereof, canbe administered alone or in combination with a second agent. The subjectcan be a patient infected or suspected to be infected with rabies virusor having a symptom of rabies virus-mediated disease (e.g., anneuropathology, encephalomyelitis, or anti-rabies immunoglobulin serumtiter). The treatment can be used to cure, heal, alleviate, relieve,alter, remedy, ameliorate, palliate, improve, or affect the infectionand the disease associated with the infection, the symptoms of thedisease, or a predisposition toward the disease. For the clinicalmanagement of rabies virus infection, “treatment” is frequentlyunderstood to mean the prophylaxis or prevention of a productiveinfection before the onset of illness.

An amount of an anti-rabies virus antibody effective to treat a rabiesvirus infection, or a “therapeutically effective amount” is an amount ofthe antibody that is effective, upon single or multiple doseadministration to a subject, in inhibiting rabies virus infection,disease, or sequelae thereof, in a subject. A therapeutically effectiveamount of the antibody or antibody fragment may vary according tofactors such as the disease state, wound site, rabies virus strain orisolate, animal vector of rabies virus, age, sex, and weight of theindividual, and the ability of the antibody or antibody portion toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody or antibody portion is outweighed by the therapeuticallybeneficial effects. The ability of an antibody to inhibit a measurableparameter can be evaluated in an animal model system predictive ofefficacy in humans. For example, the ability of an anti-rabies virusantibody to protect hamsters from lethal challenge with rabies virus canpredict efficacy in humans, as described in the Examples. Alternatively,this property of an antibody or antibody composition can be evaluated byexamining the ability of the compound to modulate rabies virus/cellinteractions, e.g., binding, infection, virulence, and the like, by invitro by assays known to the skilled practitioner. In vitro assaysinclude binding assays, such as ELISA, and neutralization assays.

An amount of an anti-rabies virus antibody effective to prevent adisorder, or a “prophylactically effective amount,” of the antibody isan amount that is effective, upon single- or multiple-doseadministration to the subject, in preventing or delaying the occurrenceof the onset or recurrence of rabies virus, or inhibiting a symptomthereof. However, if longer time intervals of protection are desired,increased doses or more frequent doses can be administered.

The terms “antagonize”, “induce”, “inhibit”, “potentiate”, “elevate”,“increase”, “decrease”, or the like, e.g., which denote quantitativedifferences between two states, refer to a difference, e.g., astatistically or clinically significant difference, between the twostates.

The term “specific binding” or “specifically binds to” refers to theability of an antibody to bind to a rabies virus, or portion thereof,with an affinity of at least 1×10⁻⁶ M, and/or bind to a rabies virus, orportion thereof, with an affinity that is at least two-fold greater thanits affinity for a nonspecific antigen.

An “antibody” is a protein including at least one or two, heavy (H)chain variable regions (abbreviated herein as VH), and at least one ortwo light (L) chain variable regions (abbreviated herein as VL). The VHand VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (CDRs),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. Sequences of Proteins ofImmunological Interest, Fifth

Edition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242, 1991, and Chothia, C. et al., J. Mol. Biol. 196:901-917,1987, which are incorporated herein by reference). Preferably, each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

The VH or VL regions of the antibody can further include all or part ofa heavy or light chain constant region. In one embodiment, the antibodyis a tetramer of two heavy immunoglobulin chains and two lightimmunoglobulin chains, wherein the heavy and light immunoglobulin chainsare inter-connected by, e.g., disulfide bonds. The heavy chain constantregion includes three domains, CH1, CH2 and CH3. The light chainconstant region is comprised of one domain, CL. The variable region ofthe heavy and light chains contains a binding domain that interacts withan antigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. The term “antibody”includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (aswell as subtypes thereof), wherein the light chains of theimmunoglobulin may be of types kappa or lambda.

The term “immunoglobulin” refers to a protein consisting of one or morepolypeptides substantially encoded by immunoglobulin genes. Therecognized human immunoglobulin genes include the kappa, lambda, alpha(IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon(IgE), and mu (IgM) constant region genes, as well as the myriadimmunoglobulin variable region genes. Full-length immunoglobulin “lightchains” (about 25 K_(D) and 214 amino acids) are encoded by a variableregion gene at the NH₂-terminus (about 110 amino acids) and a kappa orlambda constant region gene at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 K_(D) and 446 amino acids), aresimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids). The term “immunoglobulin” includes animmunoglobulin having: CDRs from a human or non-human source. Theframework of the immunoglobulin can be human, humanized, or non-human,e.g., a murine framework modified to decrease antigenicity in humans, ora synthetic framework, e.g., a consensus sequence. A matureimmunoglobulin/antibody variable region is typically devoid of a leadersequence. Immunoglobulins/antibodies can be further distinguished bytheir constant regions into class (e.g., IgA, IgD, IgE, IgG, or IgM) andsubclass or isotype (e.g., IgG1, IgG2, IgG3, or IgG4).

The term “antigen binding portion” of an antibody (or simply “antibodyportion,” or “portion”), as used herein, refers to a portion of anantibody that specifically binds to a rabies virus or component thereof(e.g., G glycoprotein), e.g., a molecule in which one or moreimmunoglobulin chains is not full length, but which specifically bindsto a rabies virus or component thereof. Examples of binding portionsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab')₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., Nature 341:544-546, 1989),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR) having sufficient framework to specificallybind, e.g., an antigen binding portion of a variable region. An antigenbinding portion of a light chain variable region and an antigen bindingportion of a heavy chain variable region, e.g., the two domains of theFv fragment, VL and VH, can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules (knownas single chain Fv (scFv); see e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also encompassed withinthe term “antigen binding portion” of an antibody. These antibodyportions are obtained using conventional techniques known to those withskill in the art, and the portions are screened for utility in the samemanner as are intact antibodies.

The term “monospecific antibody” refers to an antibody that displays asingle binding specificity and affinity for a particular target, e.g.,epitope. This term includes a “monoclonal antibody” or “monoclonalantibody composition,” which as used herein refer to a preparation ofantibodies or portions thereof with a single molecular composition.

The term “recombinant” antibody, as used herein, refers to antibodiesthat are prepared, expressed, created, or isolated by recombinant means,such as antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes orantibodies prepared, expressed, created, or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant antibodies include humanized, CDRgrafted, chimeric, in vitro generated (e.g., by phage display)antibodies, and may optionally include constant regions derived fromhuman germline immunoglobulin sequences.

The term “substantially identical” (or “substantially homologous”)refers to a first amino acid or nucleotide sequence that contains asufficient number of identical or equivalent (e.g., with a similar sidechain, e.g., conserved amino acid substitutions) amino acid residues ornucleotides to a second amino acid or nucleotide sequence such that thefirst and second amino acid or nucleotide sequences have similaractivities. In the case of antibodies, the second antibody has the samespecificity and has at least 50% of the affinity of the first antibody.

Calculations of “homology” between two sequences are performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 50% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. The percent homology between two amino acid sequences isdetermined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453,1970, algorithm which has been incorporated into the GAP program in theGCG software package, using a Blossum 62 scoring matrix with a gappenalty of 12, a gap extend penalty of 4, and a frame shift gap penaltyof 5.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which isincorporated herein by reference. Aqueous and nonaqueous methods aredescribed in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: 1) lowstringency hybridization conditions: 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); 2) medium stringency hybridizationconditions: 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridizationconditions: 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and 4) very high stringency hybridizationconditions: 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one ormore washes at 0.2×SSC, 1% SDS at 65° C.

It is understood that the antibodies and antigen binding portionsthereof described herein may have additional conservative ornon-essential amino acid substitutions, which do not have a substantialeffect on the polypeptide functions. Whether or not a particularsubstitution will be tolerated, i.e., will not adversely affect desiredbiological properties, such as binding activity, can be determined asdescribed in Bowie et al., Science, 247:1306-1310, 1990. A “conservativeamino acid substitution” is one in which an amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide, such as a binding agent,e.g., an antibody, without substantially altering a biological activity,whereas an “essential” amino acid residue results in such a change.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Overview

Rabies virus is a RNA virus that causes fatal encephalitis in humans.Provided herein are methods and compositions for treatment andprevention of rabies virus infected animals, in particular, humansubjects, more particularly, humans in need of post exposure rabiestreatment or post exposure prophylaxis (PEP). The compositions includeantibodies that recognize proteins and other molecular components (e.g.,lipids, carbohydrates, nucleic acids) of rabies virus, includingantibodies that recognize the rabies virus G glycoprotein, or portionthereof. In particular, recombinant fully human monoclonal antibodiesare provided. In certain embodiments, these human monoclonal antibodiesare produced in mice expressing human immunoglobulin gene segments(described below). Combinations of anti-rabies virus antibodies are alsoprovided.

The new methods include administering antibodies (and antigen-bindingportions thereof) that bind to rabies virus in a subject to inhibitrabies virus-mediated disease in the subject. For example, humanmonoclonal anti-rabies virus antibodies described herein can neutralizerabies virus and inhibit end-stage rabies infection and encephalitis. Inother examples, combinations of anti-rabies virus antibodies (e.g.,anti-rabies virus G glycoprotein monoclonal antibodies) can beadministered to inhibit rabies virus-mediated disease. The humanmonoclonal antibodies can be locally administered (infiltrated) at thewound site of rabies infection and, optionally, followed byadministration of an anti-rabies vaccine.

1. Generation of Antibodies

Immunogens

In general, animals are immunized with virus and/or antigens expressedby rabies virus to produce antibodies. For producing anti-rabies virusantibodies, animals are typically immunized with inactivated rabiesvirus. Rabies virus can be inactivated, e.g., by chemical treatmentand/or lyophilization and several rabies virus vaccines are availablecommercially.

Anti-rabies virus antibodies that bind and neutralize rabies virus caninteract with specific epitopes of rabies virus, for example, rabiesvirus G glycoprotein. For example, an anti-rabies virus G glycoproteincan bind an epitope within a N-terminal region of the rabies virus Gglycoprotein, or a C-terminal region, or an internal region of theprotein or fragment thereof (see Example 4 and FIG. 5) or a combinationthereof. In one example, an antibody that binds and neutralizes rabiesvirus binds to an epitope, for example, a linear epitope, within aminoacids 19-422 of rabies virus G glycoprotein. In another example, anantibody is identified that binds a linear epitope and/or conformationalepitope within amino acids 19-422 of rabies virus G glycoprotein. Asdiscussed herein, such epitopes can also be used to identify otherantibodies that bind rabies.

Generation of Human Monoclonal Antibodies in HuMAb Mice

Monoclonal antibodies can be produced in a manner not possible withpolyclonal antibodies. Polyclonal antisera vary from animal to animal,whereas monoclonal preparations exhibit a uniform antigenic specificity.Murine animal systems are useful to generate monoclonal antibodies, andimmunization protocols, techniques for isolating and fusing splenocytes,and methods and reagents for producing hybridomas are well known.Monoclonal antibodies can be produced by a variety of techniques,including conventional monoclonal antibody methodology, e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,Nature, 256: 495, 1975. See generally, Harlow, E. and Lane, D.Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988.

Although these standard techniques are known, it is desirable to usehumanized or human antibodies rather than murine antibodies to treathuman subjects, because humans mount an immune response to antibodiesfrom mice and other species. The immune response to murine antibodies iscalled a human anti-mouse antibody or HAMA response (Schroff, R. et al.,Cancer Res., 45, 879-885, 1985) and is a condition that causes serumsickness in humans and results in rapid clearance of the murineantibodies from an individual's circulation. The immune response inhumans has been shown to be against both the variable and the constantregions of murine immunoglobulins. Human monoclonal antibodies are saferfor administration to humans than antibodies derived from other animalsand human polyclonal antibodies.

One useful type of animal in which to generate human monoclonalantibodies is a transgenic mouse that expresses human immunoglobulingenes rather than its own mouse immunoglobulin genes. Such transgenicmice, e.g., “HuMAb™” mice, contain human immunoglobulin gene minilocithat encode unrearranged human heavy (μ and γ) and κ light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, N. etal., Nature 368(6474): 856-859, 1994, and U.S. Pat. No. 5,770,429).Accordingly, the mice exhibit reduced expression of mouse IgM or κ, andin response to immunization, the introduced human heavy and light chaintransgenes undergo class switching and somatic mutation to generate highaffinity human IgGκ monoclonal antibodies (Lonberg, N. et al., supra;reviewed in Lonberg, N. Handbook of Experimental Pharmacology113:49-101, 1994; Lonberg, N. and Huszar, D. , Intern. Rev. Immunol.,13: 65-93, 1995, and Harding, F. and Lonberg, N. , Ann. N.Y. Acad. Sci.,764:536-546, 1995).

The preparation of such transgenic mice is described in further detailin Taylor, L. et al., Nucleic Acids Research, 20:6287-6295, 1992; Chen,J. et al., International Immunology 5: 647-656, 1993; Tuaillon et al.,Proc. Natl. Acad. Sci., USA 90:3720-3724, 1993; Choi et al., NatureGenetics, 4:117-123, 1993; Chen, J. et al., EMBO J. ,12: 821-830, 1993;Tuaillon et al., J. Immunol., 152:2912-2920, 1994; Taylor, L. et al.,International Immunology, 6: 579-591, 1994; and Fishwild, D. et al.,Nature Biotechnology, 14: 845-851, 1996. See further, U.S. Pat. Nos.5,545,806; 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,874,299 and 5,877,397, all by Lonberg and Kay,and PCT Publication Nos. WO 01/14424, WO 98/24884, WO 94/25585, WO93/1227, and WO 92/03918.

To generate fully human monoclonal antibodies to an antigen, HuMAb micecan be immunized with an immunogen, as described by Lonberg, N. et al.Nature, 368(6474): 856-859, 1994; Fishwild, D. et al ., NatureBiotechnology, 14: 845-851, 1996 and WO 98/24884. Preferably, the miceare 6-16 weeks of age upon the first immunization. For example, apurified preparation of inactivated rabies virus can be used to immunizethe HuMAb mice intraperitonealy. To generate antibodies against rabiesvirus proteins, lipids, and/or carbohydrate molecules, mice can beimmunized with live, killed or nonviable inactivated and/or lyophilizedrabies virus. In another embodiment, a rabies virus G glycoprotein, orone or more fragments thereof, can be used as an immunogen.

HuMAb transgenic mice respond best when initially immunizedintraperitoneally (IP) with antigen in complete Freund's adjuvant,followed by IP immunizations every other week (up to a total of 6) withantigen in incomplete Freund's adjuvant. The immune response can bemonitored over the course of the immunization protocol with plasmasamples being obtained by retroorbital bleeds. The plasma can bescreened, for example by ELISA or flow cytometry, and mice withsufficient titers of anti-rabies virus human immunoglobulin can be usedfor fusions. Mice can be boosted intravenously with antigen 3 daysbefore sacrifice and removal of the spleen. It is expected that multiplefusions for each antigen may need to be performed. Several mice aretypically immunized for each antigen.

The mouse splenocytes can be isolated and fused with PEG to a mousemyeloma cell line based upon standard protocols. The resultinghybridomas are then screened for the production of antigen-specificantibodies. For example, single cell suspensions of spleenic lymphocytesfrom immunized mice are fused to one-sixth the number of P3X63-Ag8.653nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cellsare plated at approximately 2×10⁵ in flat bottom microtiter plate,followed by a two week incubation in selective medium containing 20%fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mML-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1× HAT (Sigma; the HAT is added 24 hours after thefusion). After two weeks, cells are cultured in medium in which the HATis replaced with HT. Supernatants from individual wells are thenscreened by ELISA for human anti-rabies virus monoclonal IgM and IgGantibodies. The antibody secreting hybridomas are replated, screenedagain, and if still positive for human IgG, anti-rabies virus monoclonalantibodies, can be subcloned at least twice by limiting dilution. Thestable subclones are then cultured in vitro to generate small amounts ofantibody in tissue culture medium for characterization.

In one embodiment, the transgenic animal used to generate humanantibodies to the rabies virus contains at least one, typically 2-10,and sometimes 25-50 or more copies of the transgene described in Example12 of WO 98/24884 (e.g., pHC1 or pHC2) bred with an animal containing asingle copy of a light chain transgene described in Examples 5, 6, 8, or14 of WO 98/24884, and the offspring bred with the J_(H) deleted animaldescribed in Example 10 of WO 98/24884, the contents of which are herebyexpressly incorporated by reference. Animals are bred to homozygosityfor each of these three traits. Such animals have the followinggenotype: a single copy (per haploid set of chromosomes) of a humanheavy chain unrearranged mini-locus (described in Example 12 of WO98/24884), a single copy (per haploid set of chromosomes) of arearranged human K light chain construct (described in Example 14 of WO98/24884), and a deletion at each endogenous mouse heavy chain locusthat removes all of the functional J_(H) segments (described in Example10 of WO 98/24884). Such animals are bred with mice that are homozygousfor the deletion of the J_(H) segments (Examples 10 of WO 98/24884) toproduce offspring that are homozygous for the J_(H) deletion andhemizygous for the human heavy and light chain constructs. The resultantanimals are injected with antigens and used for production of humanmonoclonal antibodies against these antigens.

The B cells isolated from such an animal are monospecific with regard tothe human heavy and light chains because they contain only a single copyof each gene. Furthermore, they will be monospecific with regard tohuman or mouse heavy chains because both endogenous mouse heavy chaingene copies are nonfunctional by virtue of the deletion spanning theJ_(H) region introduced as described in Examples 9 and 12 of WO98/24884. Furthermore, a substantial fraction of the B cells will bemonospecific with regards to the human or mouse light chains, becauseexpression of the single copy of the rearranged human kappa light chaingene will allelically and isotypically exclude the rearrangement of theendogenous mouse kappa and lambda chain genes in a significant fractionof B-cells.

In one embodiment, the transgenic mouse will exhibit immunoglobulinproduction with a significant repertoire, ideally substantially similarto that of a native mouse. Thus, for example, in embodiments where theendogenous Ig genes have been inactivated, the total immunoglobulinlevels will range from about 0.1 to 10 mg/ml of serum, e.g., 0.5 to 5mg/ml, or at least about 1.0 mg/ml. When a transgene capable ofeffecting a switch to IgG from IgM has been introduced into thetransgenic mouse, the adult mouse ratio of serum IgG to IgM ispreferably about 10:1. The IgG to IgM ratio will be much lower in theimmature mouse. In general, greater than about 10%, e.g., about 40 to80% of the spleen and lymph node B cells will express exclusively humanIgG protein.

The repertoire in the transgenic mouse will ideally approximate thatshown in a non-transgenic mouse, usually at least about 10% as high,preferably 25 to 50% or more as high. Generally, at least about athousand different immunoglobulins (ideally IgG), preferably 10⁴ to 10⁶or more, will be produced, depending primarily on the number ofdifferent V, J, and D regions introduced into the mouse genome.Typically, the immunoglobulins will exhibit an affinity for preselectedantigens of at least about 10⁻⁶ M, 10⁻⁷ M , 10⁻⁸ M , 10⁻⁹ M , 10⁻¹⁰ M ,10⁻¹¹ M , 10⁻¹² M , 10⁻¹³ M, 10⁻¹⁴ M, or greater, e.g., up to 10⁻¹⁵ M ormore.

HuMAb mice can produce B cells that undergo class-switching viaintratransgene switch recombination (cis-switching) and expressimmunoglobulins reactive with the rabies virus. The immunoglobulins canbe human sequence antibodies, wherein the heavy and light chainpolypeptides are encoded by human transgene sequences, which may includesequences derived by somatic mutation and V region recombinatorialjoints, as well as germline-encoded sequences. These human sequenceimmunoglobulins can be referred to as being substantially identical to apolypeptide sequence encoded by a human VL or VH gene segment and ahuman JL or JL segment, even though other non-germline sequences may bepresent as a result of somatic mutation and differential V-J and V-D-Jrecombination joints. With respect to such human sequence antibodies,the variable regions of each chain are typically at least 80 percentencoded by human germline V, J, and, in the case of heavy chains, D,gene segments. Frequently at least 85 percent of the variable regionsare encoded by human germline sequences present on the transgene. Often90 or 95 percent or more of the variable region sequences are encoded byhuman germline sequences present on the transgene. However, sincenon-germline sequences are introduced by somatic mutation and VJ and VDJjoining, the human sequence antibodies will frequently have somevariable region sequences (and less frequently constant regionsequences) that are not encoded by human V, D, or J gene segments asfound in the human transgene(s) in the germline of the mice. Typically,such non-germline sequences (or individual nucleotide positions) willcluster in or near CDRs, or in regions where somatic mutations are knownto cluster.

The human sequence antibodies that bind to the rabies virus can resultfrom isotype switching, such that human antibodies comprising a humansequence gamma chain (such as gamma 1, gamma 2, or gamma 3) and a humansequence light chain (such as K) are produced. Such isotype-switchedhuman sequence antibodies often contain one or more somatic mutation(s),typically in the variable region and often in or within about 10residues of a CDR) as a result of affinity maturation and selection of Bcells by antigen, particularly subsequent to secondary (or subsequent)antigen challenge. These high affinity human sequence antibodies havebinding affinities of at least about 1×10⁻⁹ M, typically at least 5×10⁻⁹M, frequently more than 1×10⁻¹⁰ M, and sometimes 5×10⁻¹⁰ M to 1×10⁻¹¹ Mor greater.

Anti-rabies virus antibodies can also be raised in other mammals,including non-transgenic mice, humans, rabbits, and goats.

Anti-Rabies Virus Antibodies

Human monoclonal antibodies that specifically bind to rabies virusinclude antibodies produced by the clones 17C7, 6G11, 5G5, 2B10, and 1E5described herein (referred to as, respectively, antibody clones 17C7,6G11, 5G5, 2B10, and 1E5). Antibodies with variable heavy chain andvariable light chain regions that are at least 80% identical to thevariable heavy and light chain regions of 17C7, 6G11, 5G5, 2B10, or 1E5can also bind to rabies virus. In related embodiments, anti-rabies virusantibodies include, for example, complementarity determining regions(CDRs) that are at least 80% identical to the CDRs of the variable heavychains and/or variable light chains of 17C7, 6G11, 5G5, 2B10, or 1E5.The CDRs of the variable heavy chain regions from these antibody clonesare shown in Table 1, below.

TABLE 1 Variable Heavy Chain CDR Amino Acid Sequences Ab Amino AcidSEQ ID Clone Chain CDR Sequence NO: 17C7 H CDR1 TYAMH 3 17C7 H CDR2VVSYDGRTKDYADSVKG 4 17C7 H CDR3 ERFSGAYFDY 5 6G11 H CDR1 GFTFSSYG 176G11 H CDR2 VAVIL 18 6G11 H CDR3 ARIAPAGSAFDY 19

The CDRs of the variable light chain regions from these clones are shownin Table 2, below.

TABLE 2 Variable Light Chain CDR Amino Acid Sequences Amino Acid SEQ IDClone Chain CDR Sequence NO: 17C7 L CDR1 RASQSVSSYLA 6 17C7 L CDR2DASNRAT 7 17C7 L CDR3 QQRNNWP 8 6G11 L CDR1 QGISSV 20 6G11 L CDR2 DAS 216G11 L CDR3 QQFNSYPPT 22

CDRs are the portions of immunoglobulins that determine specificity fora particular antigen. In certain embodiments, CDRs corresponding to theCDRs in Tables 1 and 2 having sequence variations (e.g., conservativesubstitutions) may bind to rabies virus. For example, CDRs, in which 1,2, 3, 4, or 5 residues, or less than 20% of total residues in the CDR,are substituted or deleted can be present in an antibody (or antigenbinding portion thereof) that binds rabies virus.

Similarly, anti-rabies virus antibodies can have CDRs containing aconsensus sequence, as sequence motifs conserved amongst multipleantibodies can be important for binding activity.

For example, the invention provides for the use of one or more CDRregions or derivatives of the disclosed CDRs. Such derivative CDRs arederived from a disclosed CDR or portion thereof and, optionally, alteredat one more amino acid positions. Alterations include one or more aminoacid additions, deletions, or substitutions as described herein.Exemplary residue positions for altering include those amino acidpositions identified as subject to more variance than other amino acidpositions, for example, positions subject to somatic mutations as knownin the art. Alternatively, such positions can be identified by comparingtwo or more sequences known to have a desired binding activity andidentifying CDR residues that vary and CDR residues that are constant.For example, a comparison of the variable regions of 17C7 and 6G11 heavyand light chains are presented below (Tables 3-4) and the CDR derivativeor consensus sequences that can be determined therefrom are shown(Tables 5-6).

TABLE 3 Comparison of Heavy Chain Variable Regions Comparison of:17c7H                                         −125 aa6G11H                                         −125 aausing matrix file: BLOSUM50, gap penalties: −14/−487.2% identity in 125 aa overlap; score: 731       10        20        30        40        50        60QVQLVESGGGVVQPGRSLRLSCAASGFTFS TYAMH WVRQAPGKGLEWVA VVSYDGRTKDY     ::::::::::::::::::::::::::::::.:.:::::::::::::::::. ::: .: .QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLEW VAVIL YDGSNKYH               10        20        30        40        50        60       70        80        90       100       110       120 ADSVKGRFTISRDNSKNTLYLQMNSLRTEDTAVYFCAR ERFSGAYFDY WGQGTLVTVSSA:::::::::::::::::::::::::::.::::::.::    .:. ::::::::::::::ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARIAPAGSAFDY WGQGTLVTVSSA       70        80        90       100       110       120STKGP (residue 20-144 of SEQ ID NO: 1) :::::STKGP (residue 20-144 of SEQ ID NO: 15)

TABLE 4 Comparison of Light Chain Variable Regions Comparison of:17c7L                                          −106 aa6G11L                                          −106 aausing matrix file: BLOSUM50, gap penalties: −14/−471.7% identity in 106 aa overlap; score: 527       10        20        30        40        50        60IVLTQSPATLSLSPGERATLSC RASQSVSSYL AWYQQKPGQAPRLLIY DASNRAT GIPAR : :::::..:: : :.:.:..:::::..:: ::::::: :.::..::::::.  .:.:.:IQLTQSPSSLSASVGDRVTITCRAS QGISSV LAWYQQKSGKAPKFLIY DAS SLESGVPSR         10        20        30        40        50        60       70        80        90       100  FSGSGSGTDFTLTISSLEPEDFAVYSCQQRNNWP PTFGGGTKVEIK (residue 22-127 of SEQ ID NO: 2):::::::::::::::::.:::::.: ::: :..::::: :::.:::FSGSGSGTDFTLTISSLQPEDFATYY CQQFNSYPPTFGQGTKLEIK (residue 22-127 of SEQ ID NO: 16)

Exemplary CDRs derivative or consensus sequences are presented below.

TABLE 5 Heavy Chain CDR Derivatives CDR Formula Modifications CDR1GFTFSX1YX2MH X can be any amino SEQ ID NO: 29_ acid or X1 = T/S;  X2 =A/G CDR2 VAVX1X2YDGX3X4KX5X6  X can be any amino ADSVKG acid orSEQ ID NO: 30 X1 = V/I; X2 = S/L; X3 = R/S; X4 = I/N;  X5 = D/Y; X6 =Y/H CDR3 ARX1X2X3GX4X5FDY X can be any amino SEQ ID NO: 31 acid or X1 =E/I; X2 = R/A; X3 = F/P; X4 = A/S;  X5 = Y/S

TABLE 6 Light Chain CDRs Derivatives CDR Formula Modifications CDR1RASQX1X2SSX3L  X can be any amino SEQ ID NO: 32 acid or X1 = S/G;  X2 =V/I; X3 = Y/V CDR2 DASX1X2X3X4 X can be any amino acid SEQ ID NO: 33or X1 = N/S1; X2 = R/L;  X3 = A/E; X4 = T/S CDR3 CQQX1NX2X3PX can be any amino acid or X1 = R/F; SEQ ID NO: 34 X2 = N/S; X3 = W/Y

It is also understood that one more of the CDRs disclosed herein(including CDR derivative or consensus sequences) can be used foridentifying naturally occurring CDRs that are suitable for binding to arabies virus epitope. The CDRs can also be combined or cross-clonedbetween variable regions, for example, light chain CDRs can beintroduced into heavy chain variable regions and heavy chain CDRs can beintroduced into light chain variable regions and screened to insure thatspecific binding is retained.

Human anti-rabies virus antibodies can include variable regions that arethe product of, or derived from, specific human immunoglobulin genes.For example, the antibodies can include a variable heavy chain regionthat is the product of, or derived from, a human VH 3-30-3 or VH3-33gene (see, e.g., Acc. No.: AJ555951, GI No.: 29836865; Acc. No.:AJ556080, GI No.: 29837087; Acc. No.: AJ556038, GI No.: 29837012, andother human VH3-33 rearranged gene segments provided in GenBank®). Theantibodies can also, or alternatively, include a light chain variableregion that is the product of, or derived from a human VκL6, VκL11,VκL13, VκL15, or VκL19. gene (see, e.g., GenBank® Acc. No.: AJ556049, GINo.: 29837033 for a partial sequence of a rearranged human VκL19 genesegment). As known in the art, and described in this section, above,variable immunoglobulin regions of recombined antibodies are derived bya process of recombination in vivo in which variability is introduced togenomic segments encoding the regions. Accordingly, variable regionsderived from a human VH or VL gene can include nucleotides that aredifferent that those in the gene found in non-lymphoid tissues. Thesenucleotide differences are typically concentrated in the CDRs.

Moreover, the above antibodies exhibit binding activity to a rabiesvirus and, in particular, to one or more rabies G glycoprotein epitopes.Such antibodies further exhibit rabies virus neutralization activity andin vivo protective efficacy against rabies sequelae as further describedbelow and in the examples.

2. Production and Modification of Antibodies

Many different forms of anti-rabies virus antibodies can be useful inthe inhibition of rabies virus-mediated disease. The antibodies can beof the various isotypes, including: IgG (e.g., IgG1, IgG2, IgG3, IgG4),IgM, IgA1, IgA2, IgD, or IgE. Preferably, the antibody is an IgGisotype, e.g., IgG1. The antibody molecules can be full-length (e.g., anIgG1, IgG2, IgG3, or IgG4 antibody) or can include only anantigen-binding fragment (e.g., a Fab, F(ab′)₂, Fv or a single chain Fvfragment). These include monoclonal antibodies (e.g., human monoclonalantibodies), recombinant antibodies, chimeric antibodies, and humanizedantibodies, as well as antigen-binding portions of the foregoing.

Anti-rabies virus antibodies or portions thereof useful in the presentinvention can also be recombinant antibodies produced by host cellstransformed with DNA encoding immunoglobulin light and heavy chains of adesired antibody. Recombinant antibodies may be produced by knowngenetic engineering techniques. For example, recombinant antibodies canbe produced by cloning a nucleotide sequence, e.g., a cDNA or genomicDNA, encoding the immunoglobulin light and heavy chains of the desiredantibody. The nucleotide sequence encoding those polypeptides is theninserted into an expression vector so that both genes are operativelylinked to their own transcriptional and translational expression controlsequences. The expression vector and expression control sequences arechosen to be compatible with the expression host cell used. Typically,both genes are inserted into the same expression vector. Prokaryotic oreukaryotic host cells may be used.

Expression in eukaryotic host cells is preferred because such cells aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active antibody. However, any antibodyproduced that is inactive due to improper folding can be renaturedaccording to well known methods (Kim and Baldwin, Ann. Rev. Biochem.,51:459-89, 1982). It is possible that the host cells will produceportions of intact antibodies, such as light chain dimers or heavy chaindimers, which also are antibody homologs according to the presentinvention.

The antibodies described herein also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(Morrison, S., Science, 229:1202, 1985). For example, in one embodiment,the gene(s) of interest, e.g., human antibody genes, can be ligated intoan expression vector such as a eukaryotic expression plasmid such asused in a GS gene expression system disclosed in WO 87/04462, WO89/01036 and EP 338 841, or in other expression systems well known inthe art. The purified plasmid with the cloned antibody genes can beintroduced in eukaryotic host cells such as CHO-cells or NS0-cells oralternatively other eukaryotic cells like a plant derived cells, fungi,or yeast cells. The method used to introduce these genes can be anymethod described in the art, such as electroporation, lipofectine,Lipofectamine, transfection (e.g., calcium chloride-mediated), orballistic transfection, in which cells are bombarded with microparticlescarrying the DNA of interest (Rodin, et al. Immunol. Lett.,74(3):197-200, 2000). After introducing these antibody genes in the hostcells, cells expressing the antibody can be identified and selected.These cells represent the transfectomas which can then be amplified fortheir expression level and upscaled to produce antibodies. Recombinantantibodies can be isolated and purified from these culture supernatantsand/or cells using standard techniques.

It will be understood that variations on the above procedures are usefulin the present invention. For example, it may be desired to transform ahost cell with DNA encoding either the light chain or the heavy chain(but not both) of an antibody. Recombinant DNA technology may also beused to remove some or all of the DNA encoding either or both of thelight and heavy chains that is not necessary for binding, e.g., theconstant region may be modified by, for example, deleting specific aminoacids. The molecules expressed from such truncated DNA molecules areuseful in the methods described herein. In addition, bifunctionalantibodies can be produced in which one heavy and one light chain bindto a rabies virus, and the other heavy and light chain are specific foran antigen other than the rabies virus, or another epitope of the rabiesvirus.

Also within the scope of the invention are antibodies in which specificamino acids have been substituted, deleted, or added. In particular,preferred antibodies have amino acid substitutions in the frameworkregion, such as to improve binding to the antigen. For example, aselected, small number of acceptor framework residues of theimmunoglobulin chain can be replaced by the corresponding donor aminoacids. Preferred locations of the substitutions include amino acidresidues adjacent to the CDR, or which are capable of interacting with aCDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting aminoacids from the donor are described in U.S. Pat. No. 5,585,089 (e.g.,columns 12-16), the contents of which are hereby incorporated byreference. The acceptor framework can be a mature human antibodyframework sequence or a consensus sequence. As desired, the Fc region ofantibodies of the invention can be altered to modulate effectorfunction(s) such as, for example, complement binding and/or Fc receptorbinding. Criteria and subsets of framework alterations and/or constantregions suitable for alteration (by, e.g., substitution, deletion, orinsertion) are described in U.S. Pat. Nos. 6,548,640; 5,859,205;6,632,927; 6,407,213; 6,054,297; 6,639,055; 6,737,056; and 6,673,580.

A “consensus sequence” is a sequence formed from the most frequentlyoccurring amino acids (or nucleotides) in a family of related sequences(See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft,Weinheim, Germany 1987). In a family of proteins, each position in theconsensus sequence is occupied by the amino acid occurring mostfrequently at that position in the family. If two amino acids occurequally frequently, either can be included in the consensus sequence. A“consensus framework” of an immunoglobulin refers to a framework regionin the consensus immunoglobulin sequence.

An anti-rabies virus antibody, or antigen-binding portion thereof, canbe derivatized or linked to another functional molecule (e.g., anotherpeptide or protein). For example, an antibody can be functionally linked(by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherantibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent,and/or a protein or peptide that can mediate association with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody (or fragment thereof) is produced bycros slinking two or more of such proteins (of the same type or ofdifferent types). Suitable crosslinkers include those that areheterobifunctional, having two distinct reactive groups separated by anappropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester)or homobifunctional (e.g., disuccinimidyl suberate). Such linkers areavailable from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which a antibody (or fragment thereof) canbe derivatized (or labeled) include fluorescent compounds, variousenzymes, prosthetic groups, luminescent materials, bioluminescentmaterials, and radioactive materials.

Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, and, phycoerythrin. A protein or antibody canalso be derivatized with detectable enzymes, such as alkalinephosphatase, horseradish peroxidase, β-galactosidase,acetylcholinesterase, glucose oxidase and the like. When a protein isderivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. A protein can also be derivatized with a prosthetic group(e.g., streptavidin/biotin and avidin/biotin). For example, an antibodycan be derivatized with biotin, and detected through indirectmeasurement of avidin or streptavidin binding.

Labeled proteins and antibodies can be used, for example, diagnosticallyand/or experimentally in a number of contexts, including (i) to isolatea predetermined antigen by standard techniques, such as affinitychromatography or immunoprecipitation; and (ii) to detect apredetermined antigen (e.g., a rabies virus, or rabies virus protein,carbohydrate, or lipid, or combination thereof, e.g., in a cellularlysate or a patient sample) in order to monitor virus and/or proteinlevels in tissue as part of a clinical testing procedure, e.g., todetermine the efficacy of a given treatment regimen.

Any of the above protein derivatizing/labeling techniques can also beemployed on a viral target, for example, a rabies protein, such as a Gglycoprotein or fragment(s) thereof.

3. Screening Methods

Anti-rabies virus antibodies can be characterized for binding to therabies virus by a variety of known techniques. Antibodies are typicallycharacterized by ELISA first. Briefly, microtiter plates can be coatedwith the target antigen in PBS, for example, the rabies virus or Gglycoprotein or portion thereof, and then blocked with irrelevantproteins such as bovine serum albumin (BSA) diluted in PBS. Dilutions ofplasma from mice immunized with the target antigen, for example, arabies vaccine, are added to each well and incubated for 1-2 hours at37° C. The plates are washed with PBS/Tween 20 and then incubated with agoat-anti-human IgG Fc-specific polyclonal reagent conjugated toalkaline phosphatase for 1 hour at 37° C. After washing, the plates aredeveloped with ABTS substrate, and analyzed at OD of 405. Preferably,mice which develop the highest titers will be used for fusions.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with rabies virus. Hybridomas that produce antibodies thatbind, preferably with high affinity, to rabies virus can than besubcloned and further characterized. One clone from each hybridoma,which retains the reactivity of the parent cells (by ELISA), can then bechosen for making a cell bank, and for antibody purification.

To purify the anti-rabies virus antibodies, selected hybridomas can begrown in roller bottles, two-liter spinner-flasks or other culturesystems. Supernatants can be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.) topurify the protein. After buffer exchange to PBS, the concentration canbe determined by spectrophotometric methods.

To determine if the selected monoclonal antibodies bind to uniqueepitopes, each antibody can be biotinylated using commercially availablereagents (Pierce, Rockford, Ill.). Biotinylated MAb binding can bedetected with a streptavidin labeled probe. Anti-rabies virus antibodiescan be further tested for reactivity with the rabies virus or rabiesvirus protein by immunoprecipitation or immunoblot.

Particular antibodies of the invention are characterized as binding toone or more epitope of a rabies G glycoprotein. For example, the rabiesG glycoprotein epitope can be a linear epitope, conformational epitope,discontinuous epitope, or combinations of such epitopes.

In one embodiment, the rabies G glycoprotein epitope consists ofantigenic site I, antigenic site II, antigenic site III, antigenic siteminor A, or combinations of such antigenic sites, for example, antigenicsite III and minor site A.

In another embodiment, the epitope of the rabies G glycoproteincomprises amino acid residues 336-442. In a particular embodiment, therabies G glycoprotein comprises amino acid residue 336 and, optionally,alterations thereof such as substitutions or deletions (e.g., see Table9).

Other assays to measure activity of the anti-rabies virus antibodiesinclude neutralization assays. In vitro neutralization assays canmeasure the ability of an antibody to inhibit a cytopathic effect,infectivity, or presence of a virus on or in cells in culture (seeExample 3, below). In vivo neutralization or survival assays can be usedto measure rabies virus neutralization as a function of reducedmorbidity and/or mortality in an appropriate animal model (see Examples5, below).

4. Pharmaceutical Compositions and Kits

In another aspect, the present invention provides compositions, e.g.,pharmaceutically acceptable compositions, which include an antibodymolecule described herein or antigen binding portion thereof, formulatedtogether with a pharmaceutically acceptable carrier.

“Pharmaceutically acceptable carriers” include any and all solvents,dispersion media, isotonic and absorption delaying agents, and the likethat are physiologically compatible. The carriers can be suitable forintravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, orepidermal administration (e.g., by injection or infusion).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, liposomes and suppositories. The preferred form dependson the intended mode of administration and therapeutic application.Useful compositions are in the form of injectable or infusiblesolutions. A useful mode of administration is parenteral (e.g.,intravenous, subcutaneous, intraperitoneal, intramuscular). For example,the antibody or antigen binding portion thereof can be administered byintravenous infusion or injection. In another embodiment, the antibodyor antigen binding portion thereof is administered by intramuscular orsubcutaneous injection.

The composition of the invention may be co-administered with a) one ormore other antibodies, e.g., anti-rabies antibodies, b) rabies protein,e.g., a rabies vaccine, c) toxin(s) d) other therapeutic agent(s) (e.g.,antivirals), and/or e) label(s).

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intracranial,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural, andintrasternal injection and infusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high antibody concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, theuseful methods of preparation are vacuum drying and freeze-drying thatyields a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody portions described herein can beadministered by a variety of methods known in the art, and for manytherapeutic applications. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results.

In certain embodiments, an antibody, or antibody portion thereof may beorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation. Therapeutic compositions can be administered with medicaldevices known in the art.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time, orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antigen bindingportion of the invention is 0.1-60 mg/kg, e.g., 0.5-25 mg/kg, 1-2 mg/kg,or 0.75-10 mg/kg. In one embodiment, the amount of anti-rabies virusantibody (or antigen binding portion thereof) administered, is at orabout 0.125 mg/kg, 0.25 mg/kg, 0.5 mg/kg, or at an interval or rangethereof. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

Also within the scope of the invention are kits including an anti-rabiesvirus antibody or antigen binding portion thereof. The kits can includeone or more other elements including: instructions for use; otherreagents, e.g., a label, a therapeutic agent, or an agent useful forchelating, or otherwise coupling, an antibody to a label or therapeuticagent, or other materials for preparing the antibody for administration;pharmaceutically acceptable carriers; and devices or other materials foradministration to a subject.

Various combinations of antibodies can be packaged together. Forexample, a kit can include antibodies that bind to rabies virus (e.g.,antibodies that include the variable heavy and/or light chain regions of17C7, 6G11, 5G5, 2B10, E5, or a combination thereof. The antibodies canbe mixed together, or packaged separately within the kit.

Instructions for use can include instructions for therapeuticapplication including suggested dosages and/or modes of administration,e.g., in a patient with a symptom or indication of rabies virus exposureor suspected of rabies virus exposure. Other instructions can includeinstructions on coupling of the antibody to a chelator, a label or atherapeutic agent, or for purification of a conjugated antibody, e.g.,from unreacted conjugation components.

The kit can include a detectable label, a therapeutic agent, and/or areagent useful for chelating or otherwise coupling a label ortherapeutic agent to the antibody. Coupling agents include agents suchas N-hydroxysuccinimide (NHS). In such cases the kit can include one ormore of a reaction vessel to carry out the reaction or a separationdevice, e.g., a chromatographic column, for use in separating thefinished product from starting materials or reaction intermediates.

The kit can further contain at least one additional reagent, such as adiagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agentas described herein, and/or one or more additional anti-rabies virusantibodies (or portions thereof), formulated as appropriate, in one ormore separate pharmaceutical preparations.

Other kits can include optimized nucleic acids encoding anti-rabiesvirus antibodies, for use as passive immunotherapy, and/or rabies virusprotein(s), or fragments thereof, for use as, e.g., vaccines (activeimmunotherapy), and instructions for expression of the nucleic acids.

5. Therapeutic Methods and Compositions

Antibodies and antibody binding fragments of the present invention havein vitro and in vivo therapeutic, prophylactic, and diagnosticutilities. For example, these antibodies can be administered to cells inculture, e.g., in vitro or ex vivo, or in vivo, to an animal, preferablya human subject, to treat, inhibit, prevent relapse, and/or diagnoserabies virus and disease associated with rabies.

As used herein, the term “subject” is intended to include human andnon-human animals. The term “non-human animals” includes allvertebrates, e.g., mammals and non-mammals, such as non-human primates,mice, dogs, cats, pigs, cows, and horses.

The proteins and antibodies can be used on cells in culture, e.g., invitro or ex vivo. For example, cells can be cultured in vitro in culturemedium and the contacting step can be effected by adding the anti-rabiesvirus antibody or fragment thereof, to the culture medium. The methodscan be performed on virions or cells present in a subject, as part of anin vivo (e.g., therapeutic or prophylactic) protocol. For in vivoembodiments, the contacting step is effected in a subject and includesadministering an anti-rabies virus antibody or portion thereof to thesubject under conditions effective to permit binding of the antibody, orportion, to a rabies virus or any portion thereof present in thesubject, e.g., in or around a wound or on or near cells of neuronalorigin.

Methods of administering antibody molecules are described herein.Suitable dosages of the molecules used will depend on the age and weightof the subject and the particular drug used. The antibody molecules canbe used as competitive agents for ligand binding to inhibit or reduce anundesirable interaction, e.g., to inhibit binding and/or infection ofrabies virus of cells, e.g., neuronal cells.

The anti-rabies virus antibodies (or antigen binding portions thereof)can be administered in combination with other anti-rabies virusantibodies (e.g., other monoclonal antibodies, polyclonalgamma-globulin, e.g., human serum comprising anti-rabiesimmunoglobulins). Combinations of antibodies that can be used include ananti-rabies virus antibody or antigen binding portion thereof and/or ananti-rabies virus G protein antibody or antigen binding portion thereof.The anti-rabies virus or G protein antibody can be antibody clone 17C7,6G11, 5G5, 2B10, and/or E5 that includes the variable regions of such anantibody or antibodies, or an antibody with variable regions at least90% identical to the variable regions of such an antibody or antibodies.In one embodiment, the anti-rabies virus antibody can be 17C7 or portionthereof or an antibody with variable regions at least 90% identical tothe variable regions of the foregoing, e.g., 17C7, 6G11, 5G5, 2B10,and/or E5. Combinations of anti-rabies virus antibodies (e.g., 17C7,6G11, 5G5, 2B10, and/or E5) can provide potent inhibition of rabies,especially, e.g., particular rabies isolates (see Tables 11-13).Characteristic rabies virus isolates for which the antibodies of theinvention are suitable for treating, detecting, diagnosing and the likeinclude, for example, CVS-11 isolate, ERA isolate, Pasteur virusisolate, gray fox (Texas) isolate, gray fox (Arizona) isolate, artic fox(Arkansas) isolate, skunk (North Central) isolate, skunk (South Central)isolate, raccoon isolate, coyote (Texas) isolate, dog (Texas) isolate,bat (Lasiurus borealis; Tennessee) isolate, bat (Eptesicus fuscus-Myotisspp.; Colorado) isolate, bat (Myotis spp.; Washington) isolate, bat(Lasiurus cinereus; Arizona) isolate, bat (Pipistrellus subflavus;Alabama) isolate, bat (Tadarida brasiliensis; Alabama) isolate, bat(Lasionycetris noctivagans; Washington) isolate, bat (Eptesicus fuscus;Pennsylvania) isolate, mongoose (New York/Puerto Rico) isolate, dog(Argentina) isolate, dog (Sonora) isolate, dog (Gabon) isolate, dog(Thai) isolate, and combinations thereof.

It is understood that any of the agents of the invention, for example,anti-rabies virus antibodies, or fragments thereof, can be combined, forexample in different ratios or amounts, for improved therapeutic effect.Indeed, the agents of the invention can be formulated as a mixture, orchemically or genetically linked using art recognized techniques therebyresulting in covalently linked antibodies (or covalently linked antibodyfragments), having anti-rabies binding properties, for example,multi-epitope binding properties to, for example, rabies virus Gglycoprotein. The combined formulation may be guided by a determinationof one or more parameters such as the affinity, avidity, or biologicalefficacy of the agent alone or in combination with another agent. Theagents of the invention can also be administered in combination withother agents that enhance access, half-life, or stability of thetherapeutic agent in targeting, clearing, and/or sequestering rabiesvirus or an antigen thereof.

Such combination therapies are preferably additive and even synergisticin their therapeutic activity, e.g., in the inhibition, prevention,infection, and/or treatment of rabies virus-related disease ordisorders. Administering such combination therapies can decrease thedosage of the therapeutic agent (e.g., antibody or antibody fragmentmixture, or cross-linked or genetically fused bispecific antibody orantibody fragment) needed to achieve the desired effect.

Immunogenic compositions that contain an immunogenically effectiveamount of a rabies virus component, for example, rabies virus Gglycoprotein, or fragments thereof, also provided by the presentinvention, and can be used in generating anti-rabies virus antibodies.Immunogenic epitopes in a rabies virus protein sequence can beidentified as described herein (see e.g. Example 4) or according tomethods known in the art, and proteins, or fragments containing thoseepitopes can be delivered by various means, in a vaccine composition.Suitable compositions can include, for example, lipopeptides (e.g.,Vitiello et al., J. Clin. Invest. 95:341 (1995)), peptide compositionsencapsulated in poly (DL-lactide-co-glycolide) (“PLG”) microspheres(see, e.g., Eldridge et al., Molec. Immunol. 28:287-94 (1991); Alonso etal., Vaccine 12:299-306 (1994); Jones et al., Vaccine 13:675-81 (1995)),peptide compositions contained in immune stimulating complexes (ISCOMS)(see, e.g., Takahashi et al., Nature 344:873-75 (1990); Hu et al., Clin.Exp. Immunol. 113:235-43 (1998)), and multiple antigen peptide systems(MAPs) (see, e.g., Tam, Proc. Natl. Acad. Sci. U.S.A. 85:5409-13 (1988);Tam, J. Immunol. Methods 196:17-32 (1996)).

Useful carriers that can be used with immunogenic compositions of theinvention are well known, and include, for example, thyroglobulin,albumins such as human serum albumin, tetanus toxoid, polyamino acidssuch as poly L-lysine, poly L-glutamic acid, influenza, hepatitis Bvirus core protein, and the like. The compositions can contain aphysiologically tolerable (i.e., acceptable) diluent such as water, orsaline, typically phosphate buffered saline. The compositions andvaccines also typically include an adjuvant. Adjuvants such asincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, oralum are examples of materials well known in the art. Additionally, CTLresponses can be primed by conjugating target antigens, for example arabies virus protein(s) (or fragments, inactive derivatives or analogsthereof) to lipids, such astripalmitoyl-S-glycerylcysteinyl-seryl-serine (P₃CSS).

The anti-rabies antibodies can be administered in combination with otheragents, such as compositions to treat rabies virus-mediated disease. Forexample, therapeutics that can be administered in combination withanti-rabies antibodies include antiviral agents, serum immunoglobulin,and/or vaccines for treating, preventing, or inhibiting rabies (forexample, vaccines such as RabAvert™ (Chiron), Rabies vaccine adsorbed(Bioport), and Imovax™ Rabies (Aventis) and/or immunoglobulins, such asBayRab™ (Bayer) and Imogam™ Rabies-HT (Aventis). The antibody can beadministered before, after, or contemporaneously with a rabies virusvaccine.

6. Other Methods

An anti-rabies antibody (e.g., monoclonal antibody) can be used toisolate rabies virus by standard techniques, such as affinitychromatography or immunoprecipitation. Moreover, an anti-rabies antibodycan be used to detect the virus (e.g., in a serum sample), e.g., toscreen samples for the presence/exposure of rabies virus. Anti-rabiesantibodies can be used diagnostically to monitor levels of the virus intissue as part of a clinical testing procedure, e.g., to, for example,determine the efficacy of a given treatment regimen. In addition, rabiesvirus epitopes, for example, G glycoprotein epitopes (linear,conformational, or combinations thereof) can be used as immunogens or astargets to identify neutralizing anti-rabies binding molecules,including, for example, human serum, polyclonal antibodies, monoclonalantibodies, or fragments thereof.

EXEMPLIFICATION

Throughout the examples, the following materials and methods were usedunless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, e.g.,antibody technology), and standard techniques in polypeptidepreparation. See, e.g., Sambrook, Fritsch and Maniatis, MolecularCloning: Cold Spring Harbor Laboratory Press (1989); AntibodyEngineering Protocols (Methods in Molecular Biology), 510, Paul, S.,Humana Pr (1996); Antibody Engineering: A Practical Approach (PracticalApproach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: ALaboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); andCurrent Protocols in Molecular Biology, eds. Ausubel et al., John Wiley& Sons (1992). See also, e.g., Smith et al., A rapid fluorescent focusinhibition test (RFFIT) for determining rabies virus-neutralizingantibody, pages 181-189 and Chapter 15 in Laboratory Techniques inRabies, 4th ed., edited by Meslin et al., Geneva : World HealthOrganization (1996)).

Mouse Immunization and Isolation of Hybridomas

HuMab mice (Medarex) are transgenic mice containing human immunoglobulingenes and inactivated mouse heavy chain genes and kappa light chaingenes. HuMab mice were typically injected with a ˜ 1/10 of a human doseof a commercially available rabies vaccine using complete Freund'sadjuvant in the first week, and RIBI adjuvant in subsequent weeks for atotal of 6-8 weeks. A rabies envelope glycoprotein ELISA was used tomeasure serum responses and animals were sacrificed when serum responseswere considered maximal. Hybridomas were generated by fusion ofsplenocytes and partner cells (P3X63Ag8.653 mouse myeloma cells) andresultant supernatants were screened for reactivity in a rabiesglycoprotein ELISA. Positive antibodies were purified from hybridomacultures by protein A Sepharose chromatography (Amersham).

RFFIT

The RFFIT assay was performed as described in the art. The rabies virusstrains, street virus isolates, and mouse neuroblastoma cells (MNA) usedwere all from the Center from Disease Control and Prevention, Atlanta,USA.

Cells and Cell Culture

HEK-293T/17 cells, obtained from the ATCC, were grown in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal bovine serumand 100IU of penicillin-streptomycin (complete medium) at 37° C. with 5%CO2. Cells were harvested in phosphate buffered saline (PBS) containing5 mM EDTA.

Cloning of Rabies Glycoproteins

The amino acid sequence of the rabies G protein (ERA strain, Genbank:AF406693) was used to design a codon-optimized version of the rabiesglycoprotein gene spanning the full length glycoprotein from amino acid1-524. The synthetic gene was cloned into pcDNA3.1Myc/His (Invitrogen)in frame with the c-Myc and 6-histidine (His) tags (SEQ ID NO:35). Theseimmunotags enabled easy purification and detection. Truncated versionsof the tagged glycoprotein-encoding genes were constructed whichcontained the entire ectodomain (20-439 a.a.). Truncations were made byPCR amplification of the desired fragments from the full lengthglycoprotein clones followed by restriction digestion and ligation intopcDNA3.1Myc/His (Invitrogen) and verified by DNA sequence analysis.

For the isolation of native genes encoding various strains of rabies Gglycoproteins, MNA cells were infected with the CVS-11, Skunk-CA,Lasirius borealis, Lasirius cinereus, and ERA rabies viruses (Center forDisease Control and Prevention, USA). RNA was extracted from infectedcells or from virions using Trizol reagent. RTPCR was performed in 2steps. First, cDNA was synthesized using the Ambion Retroscript Kit, andthe rabies glycoprotein-encoding genes were then amplified using TurboPfu (Stratagene) and rabies virus specific primers. The rabiesglycoprotein encoding genes were cloned into the mammalian expressionvector pCDNA3.1MycHis (Invitrogen) at the HindIII/Xba I sites in framewith the c-Myc and His epitope tags. Recombinant genes encoding rabiesglycoprotein mutated at residues classified as antigenic site I, II, IIIand minor site a were synthesized using site-directed mutagenesis.Overlapping primers containing the desired point mutations were used toamplify full length mutant glycoprotein genes and the pcDNA3.1Myc/Hisvector from the previously cloned codon-optimized ERA glycoprotein. ThePCR amplified DNA was digested with DpnI to remove the wild typenon-amplified starting template, transformed into bacteria, and screenedfor the intended mutation by sequencing. The full coding sequence ofeach mutant was confirmed, and the resulting constructs were cloned intopcDNA3.1Myc/His expression vectors.

Recombinant Glycoprotein Expression

All constructs were transfected into HEK-293T/17 cells usingLipofectamine 2000 (Invitrogen) as described by the manufacturer. Cellswere grown to 85% confluence in 150 mm tissue culture dishes in 15 ml ofDMEM-10% fetal calf serum (FCS). Amounts of 30 ug of DNA mixed with 75ul of Lipofectamine were added to the cells, and plates were incubatedovernight at 37° C. Media was removed and stored at 24, 48 and 72 hourspost-transfection for secreted soluble proteins or discarded formembrane bound proteins.

Recombinant Protein Purification, Immunoprecipitation and Western Blot

Rabies glycoproteins ERA20-439 and CVS-1120-439, both containing Myc andHis epitope tags, were purified from cell culture supernatant byincubation with nickel-nitrilotriacetic acid (Ni-NTA) beads(Invitrogen), followed by column filtration and protein elution using250 mM imidazole. For immunoprecipitation of full-length membrane boundglycoproteins, transfected cells were detached from the plate with PBS/5mM EDTA and solubilized in PBS, 1% CHAPS, 1× complete proteinaseinhibitor. Cellular lysates were cleared by centrifugation and incubatedwith either HuMab 17C7, or a control non-rabies HuMab, and Protein ASepharose. Immunoprecipitated proteins were resolved by SDS-PAGE forsubsequent analysis.

For immunoblot analysis, proteins were boiled in 2× Laemmli samplebuffer (+/−BME) for 5 minutes and resolved using 10 or 12% Novex gels(Invitrogen). Gels were transferred to Immobilon P (Millipore) asdescribed by the manufacturer, and immunoblot analysis was performed.Proteins were detected using the mouse anti-Myc antibody 9E10 (0.2ug/ml) (BD Pharmingen), or HuMab 17C7 (2 ug/ml) followed by horseradishperoxidase-conjugated anti-mouse or anti-human IgG (1:5000 JacksonImmuoresearch). Membranes were incubated with enhanced chemiluminescencereagent (Amersham) for 1 minute and exposed to X-Omat-AR film forvarious periods of time.

Cell Surface Staining

Cells transfected with constructs encoding full-length rabies G proteinwere harvested 48 hours post-transfection and incubated with varyingconcentration of HuMabs. Binding of the HuMabs was detected byphycoerythrin labeled anti-human IgG (Jackson) and flow cytometry wasperformed using FACScan with CellQuest software (Becton Dickinson).

ELISAs

A capture ELISA was performed on all hybridomas to identify those makinghuman IgG. ELISA plates were coated with 3 μg/ml of goat anti-humankappa light chain antibodies (Southern Biotech). Plates were washed withwash buffer (PBS, 0.05% Tween), blocked with blocking buffer (PBS, 1%BSA, 0.05% Tween), washed, and then samples were added to plate (diluted1:2-1:400 in blocking buffer). Binding was detected with goat anti-humanIgG-AP secondary antibody (Jackson ImmunoResearch), and the plates werewashed and developed with p-Nitrophenyl phosphate disodium salt at 1mg/ml in 1M diethanolamine for 20 minutes. The plates were read at 405nm.

A capture glycoprotein ELISA was used to test the interaction of HuMab17C7 with CVS-1120-439 and codon optimized ERA20-439. Plates were coatedwith 7.5 ug/ml of mouse anti-c-Myc antibody 9E10 (BD Pharmingen) orchicken anti-c-Myc antibody (Molecular Probes). Plates were incubatedwith purified glycoproteins or detergent solubilized cell lysates, andthen incubated with primary antibodies (HuMab 17C7 and mouse anti-rabiesglycoprotein R0012 (US Biological)) at 5 ug/ml. Binding was detectedwith alkaline phosphatase conjugated goat anti-human secondary (JacksonImmunoResearch), and then developed as described above.

Production of HuMab 17C7 Resistant Viruses

Mouse neuroblastoma cells were plated at 1.5×105 cells/ml well on Day 1.On Day 2 1×101 to 108 FFU/ml of CVS-11 rabies virus was incubated withIU/ml of HuMab 17C7 (133 ug/ml) at 37° C. for 1 hour. The virus/antibodymix was added to the cells and incubated for 3-12 hours at 37° C. Thevirus/antibody mix was removed from the cells and cells were washed oncewith media, followed by addition of fresh media containing IU/ml ofHuMab 17C7 for an additional 60 hours. On Day 5 the media, containingpotential HuMab 17C7 resistant virus, was removed from the slides,labeled and stored at 4° C. Slides were then stained for presence ofrabies infected cells by incubation with 1:40 dilution of Centicor FITCanti-Rabies IgG (Fujirebio Diagnostics) for 30 minutes at 36° C./0.5%CO2. The slides were then washed and examined under a fluorescentmicroscope (FITC filter) at 200× magnification. Virus taken from wellscontaining 1-5 fluorescent foci were amplified on MNA cells for 3 daysin the presence of HuMab 17C7. The amplified virus was tested for theability to infect MNA cells equivalently in the presence and absence ofHuMab 17C7. 6-well plates of MNA cells were infected with the HuMab 17C7resistant virus. RNA was extracted from virus-infected cells, reversetranscribed, and the glycoprotein-encoding sequence was PCR amplifiedwith CVS-11 glycoprotein-specific primers. The mutations inglycoprotein-encoding genes were analyzed by sequencing the entirecoding sequence.

Rabies Psuedovirus

A replication defective Env-, Vpr-HIV backbone containing the fireflyluciferase gene inserted into the nef gene, pNL4-3.Luc.R-E-, wasco-transfected with rabies glycoprotein encoding plasmids into 293Tcells. The pNL4-3.Luc.R-E-reagent was obtained through the NIH AIDSResearch and Reference Reagent Program, Division of AIDS, NIAID, andNIH. Pseudoviral particles were harvested 48-72 hours post-transfection,concentrated 30-fold using a centricon concentrator (Millipore) andfrozen at −80° C. The luciferase counts per second of the pseudoviruspreparations were determined by serial dilution of the virus followed byinfection and detection (see below). For the neutralization assaysapproximately 50,000 counts per second of pseudovirus were incubatedwith and without antibody for 1 hour at room temperature. Theantibody/virus mix was then applied to HOS cells (ATCC# CRL-1543), inthe presence of 2 ug/ml of polybrene and spinoculated for 2 hours at800G and 4° C., followed by incubation at 37° C./5% CO2. Luciferaseactivity was then assayed 72 hours post-infection using the Bright-Gloreagent (Promega), according to the manufacturer's protocol.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Generation of Anti-Rabies Virus Monoclonal Antibodies

Transgenic mice comprising human immunoglobulin genes generated asdescribed above in the section entitled “Generation of Human MonoclonalAntibodies in HuMAb Mice” and supplied by Medarex, Milpitas, Calif.,were immunized with 6 doses of a commercial rabies vaccine. The vaccinewas administered in combination with Fruend's complete adjuvant and thenboosted with additional rabies vaccine and incomplete Fruend's adjuvant.The rabies vaccine consists of whole rabies virus that has beeninactivated and lyophilized. Spleenic B cells were isolated from theimmunized animal and fused to mouse myeloma (P3X) cells. Clonalhybridomas were generated and screened by ELISA. Resultant hybridomaswere cultured and enzyme linked immunosorbent assay (ELISA) fordetection of human kappa/gamma antibody chains was used to detectcandidate human IgG antibodies for further analysis. Clones designated54.17C7; 108.6G11; 35.5G5.1E12.149; 35.2B10.1G11.3FG; and 35.1E51G1.4CBreferred to herein as, respectively, clones 17C7, 6G11, 5G5, 2B10, and1E5 were further determined to specifically bind rabies virus Gglycoprotein by an antigen specific ELISA assay. In addition, these fivehybridoma clones were selected and determined to neutralize rabiesinfection of mouse neuronal cells in a RFFIT assay against a number ofdifferent rabies isolates (see Example 3).

Accordingly, cDNAs from exemplary clones were amplified by RT-PCR frommRNA, cloned, and sequenced. One heavy chain V region consensus sequencewas found for each clone (Table 7). All five clones utilized a VH regionderived from one of two germline V region genes, but utilized differentJ sequences. The amino acid sequences of the VH and VL regions fromexemplary clones 17C7 and 6G11 are shown in FIGS. 1-2 (SEQ ID NOs: 1, 2,15, and 16). The complementarity determining regions (i.e., CDR1, CDR2,and CDR3) are indicated for the antibody heavy and light chain variableregions (SEQ ID NOs: 3-8; 17-22). DNA encoding the antigen bindingportion of each clone was cloned into a vector to be expressed as ahuman antibody for administration to humans. The nucleic acid and aminoacid sequences for the light and heavy chains of antibody clones 17C7and 6G11 are provided in the sequence listing (respectively, SEQ ID NOs:9-12 and SEQ ID NOs: 23-26).

TABLE 7 Antibody Clones and Gene Composition Clone HuMab mouse HuMabDesignation genotype 54.17C7 17C7 Female Hco12 108.6G11 6G11 Male Hco735.5G5 5G5 Male Hco7 35.1E5 1E5 Male Hco7 35.2B10 2B10 Male Hco7 HuMabVariable region light chain Variable region heavy chain 54.17C7 VK: JK:VH: D: JH: IGKV3- IGKJ4*01 IGHV3- IGHD3- IGHJ4*02 11*01 30-3*01 3*01108.6G11 VK: JK: VH: D: JH: IGKV1- IGKJ2*02 IGHV3- IGHD6- IGHJ4*02 13*0233*05 13*01 (*IMGT nomenclature used for above table)

Example 2 Binding Activity of Anti-Rabies Virus Antibodies

Binding of each antibody to rabies virus, in particular, rabies virusglycoprotein G was determined by ELISA using standard techniques. Theaffinity of the anti-rabies virus antibodies for rabies virusglycoprotein G can also be measured with a Biacore® instrument, whichdetects biomolecular binding interactions with surface plasmon resonancetechnology. Each antibody is added to protein A-coated sensor chips, andrabies virus glycoprotein G is allowed to flow over the chip to measurebinding. Binding constants ranging from a K_(D) of 1×10⁻⁶M, K_(D) of1×10⁻⁷M, K_(D) of 1×10⁻⁸M, K_(D) of 1×10⁻⁹M. K_(D) of 1×10⁻¹⁰M. K_(D) of1×10⁻¹¹M, K_(D) of 1×10⁻¹²M, and higher (or internals or rangesthereof), can be determined. Anti-rabies virus antibodies with favorablebinding constants indicate that the antibodies have affinities suitablefor use in human therapy.

The antibody 17C7, when tested on the ectodomain region of a rabies Gglycoprotein (codon-optimized), was determined by Biacore analysis tohave a binding affinity of at least 1.36E-8 M.

Example 3 Rabies Virus Neutralization by Anti-Rabies Virus Antibodies

Antibodies expressed by 17C7, 6G11, 5G5, 2B10, and 1E5 hybridomas weretested for rabies virus neutralization activity in vitro in a series ofexperiments (see Tables 8-11, below).

Specifically, rabies neutralizing activity was determined using theRapid Fluorescent Focus Inhibition Test (RFFIT), which detects rabiesvirus infection of mouse neuroblastoma cells using fluorescent-labeledantibodies. The RFFIT assay is a standardized assay that is used bymedical and public heath experts to determine the potency of a givenantibody preparation to neutralize rabies viruses (i.e., inhibit itsability to infect cells). The assay is typically performed using a fixedvirus (CVS11) but may also be done using isolates from infected animals.The assay is done by the addition of a standard amount of virus with andwithout antibody dilutions to monolayers of mouse neuroblastoma cells.The monolayers are incubated then foci of infected neuroblastoma cellsare detected using a fluorescent-labeled anti-rabies nucleoproteinmonoclonal antibody. The foci are visualized and counted usingfluorescent microscopy. Subsequent results are reported as the antibodyconcentration (dilution) where the number of microscope fields withoutfluorescent foci is 50%. All assays include a standard rabies immuneglobulin preparation (SRIG) for comparison. Anti-rabies human monoclonalantibodies 17C7, 6G11, 5G5, 2B10, and 1E5 were tested against a panel ofrabies virus isolates of public health significance from variousvertebrate animals from North America.

Table 8 shows the results of in vitro neutralization assays of selectedantibodies, as compared to current therapies (i.e., human anti-rabiesserum; “SRIG”), against a panel of rabies virus isolates. Numbersindicate the fold dilution by which an antibody can be diluted and stillexhibit a 50% neutralization activity, i.e., the ability to block rabiesvirus infection of murine neuroblastoma cells in vitro. Results for 17C7at a higher concentration against selected isolates and are shown in thelower panel.

TABLE 8 Strain Neutralization Results 17C7 5G5 1E5 2B10 supernatant 6G112 IU/ml or 2 IU/ml or 2 IU/ml or SRIG or 2 mg/ml* 1 mg/ml 1 mg/ml* 1mg/ml* 1 mg/ml* rabies virus CVS-11 145   1100 230 ≧1400*   ≧1400*  ≧1400*   ERA 85 >1400 ≧1400 230  250  230  Pasteur virus 17 >1400 110095 145  65 Raccoon, SE US 110 >1400 1300 ≧1400*   ≧1400*   ≧1400*   Grayfox, TX 54 >1400 1100 95 95 110  Gray fox, AZ 50 >1400 1300 480  480 230  Arctic Fox, AK 54 >1400 1200 1000*  800* ≧1400*   Coyote, TX95 >1400 1200 60 60 60 Dog/Coyote, TX 50 >1400 1100 60 60 75 Skunk,north central 170  200 210 230  250 270  Skunk, south central 54 >1400≧1400 1300*  ≧1400*   ≧1400*   Skunk, CA 29 >1400 800 1300*  1200* ≧1400*   Bat, Lasiurus 42   320* <5  <5*  <5*  7* borealis, TN Bat,Eptesicus fuscus- 95  625 200 70 95 60 Myotis spp., CO Bat, Myotis spp.,WA 50 >1400 700 ≧1400*   ≧1400*   ≧1400*   Bat, Lasiurus 25   270* <5 <5*  <5*  85* cinereus, AZ Bat, Pipistrellus 29  390 13 36 45 36subflavus, AL Bat, Tadarida 50 ≧1400  125 180  210  125  brasiliensis,AL Bat, Lasionycteris 42 ≧1300  36 40 25 50 noctivagans, WA Bat,Eptesicus 11 ≧1400  <5 11 16 29 fuscus, PA Mongoose, NY/ 230 ≧1400 ≧1400 320  390  250  Puerto Rico Dog, Argentina 54 ≧1400  ≧1400 1300* 1200*  1300*  Dog, Sonora 56 ≧1400  ≧1400 19 33 56 Dog, Gabon 54 ≧1400 ≧1400 45 19 50 Dog, Thai 56 ≧1400  ≧1400 17 14 40 non rabies lyysavirusLagos <5   <5* nd nd nd nd Mokola <5   <5* nd nd nd nd Duvenhage 13  <5* nd nd nd nd European bat virus 1 42   <5* nd nd nd nd European batvirus 2 40   <5* nd nd nd nd Australian bat virus 54 ≧1400* nd nd nd nd

Initially, each of the HuMAbs was screened for the ability to neutralizethe rabies virus strain CVS-11. Neutralizing HuMabs were then testedmore extensively against a broad panel of isolates of public healthsignificance from North and South America, Europe, Africa and Asia.Strikingly, HuMab 17C7 neutralized the majority of rabies virus isolatesin contrast to HuMabs 2B10 and 5G5 (Table 8). The 50% end pointneutralization titer was determined for one of the street rabiesviruses, isolated from a Skunk in California, USA (Skunk-CA). The titercalculated for HuMab17C7 (concentration tested was 0.03mg/ml) againstCalifornia Skunk was 1:12,898, which demonstrates that HuMab 17C7potently neutralizes this street virus.

To better understand how the potency of a single human monoclonalantibody compares to polyclonal hRIG, HuMab 17C7 and hRIG were tested atidentical antibody concentrations in a RFFIT assay using the CVS-11rabies virus. The 50% endpoint titer for hRIG was 1:224, while it was1:7029 for HuMab 17C7 (FIG. 4). Therefore, HuMab 17C7 inhibitedinfection by CVS-11 more potently than hRIG at equivalent antibodyconcentrations. These initial experiments revealed that HuMab 17C7 wasable to neutralize many isolates of rabies virus, and that the extent ofneutralization ranged from the potent neutralization of the Skunk, CAisolate at a low antibody dose (0.03 ug/ml; 1:12,898) as compared to theless potent neutralization of CVS-11 at higher antibody dose (2 mg/ml;1:7029).

Repeat testing was done using purified 17C7 at varying concentrationsagainst rabies isolates that did not initially show neutralization inRFFIT testing on hybridoma supernatants (Lasiurus borealis, Tennesseeand Lasiurus cinereus, Arizona) and demonstrated to be capable ofneutralizing both viruses in the repeat assay. These data imply thatHuMab 17C7 interacts with a neutralizing epitope on the rabiesglycoprotein from the L. borealis-Tennessee and L. cinereus-Arizonaisolates (Table 9).

TABLE 9 50% End Point Neutralization (Reciprocal Titer) of HuMabs 2B10,17C7 and 5G5 in RFFITs Against Rabies Virus Isolated from North AmericanBats (Lasirius borealis and cinereus) hRIG 17C7 2B10 5G5 Rabies Isolate(2 IU/ml) (2 mg/ml) (1.5 mg/ml) (1 mg/ml) Bat, Lasiurus borealis, 42 3207 <5 TN Bat, Lasiurus 25 270 85 <5 cinereus, AZ

The HuMab 17C7 clone was also tested for its ability to neutralizenon-rabies lyssaviruses. Lyssaviruses are not a significant world-widepublic health problem, but have caused fatal disease in a small numberof human cases. These occurrences, as well as the prevalence of somelyssaviruses in wild-life reservoirs, have led to a recent interest inwhether rabies biologics protect against non-rabies lyssaviruses. HuMab17C7 was able to potently neutralize Australian bat lyssavirus whentested in a modified RFFIT assay. The titer calculated for HuMabl7C7(concentration tested was 2 mg/ml) against Australian bat lyssavirus wasgreater than 1:1400 which demonstrates that HuMab 17C7 neutralizes theAustralian bat lyssavirus (Table 10).

TABLE 10 50% End Point Neutralization (Reciprocal Titer) of HuMab 17C7in RFFITs Against Lyssaviruses. hRIG HuMab 17C7 Lyssavirus (2 IU/ml) (2mg/ml) Rabies (CVS-11) 270 >1400 Lagos <5 <5 Mokola <5 <5 Duvenhage 13<5 European bat lyssavirus 1 42 <5 European bat lyssavirus 2 40 <5Australian bat lyssavirus 54 >1400

These data show that that the anti-rabies monoclonal antibodies werecapable of neutralizing rabies virus isolates from a variety of NorthAmerican vertebrate animals of public health significance in the RFFITassay.

Example 4 Epitope Mapping of Anti-Rabies Virus G Glvcoprotein Antibodies

The epitope of rabies virus glycoprotein G bound by each monoclonalantibody was determined by immunoblotting and immunoprecipitation assays(see FIG. 3).

A full-length synthetic human codon-optimized rabies virus Gglycoprotein gene from the ERA rabies virus isolate was constructedusing polymerase chain reaction (PCR) and genetic engineering. The geneand deletion derivatives were cloned into pCDNA3.1A (Invitrogen) forexpression in human 293T cells. Immunoblot and immunoprecipitationexperiments were carried out using standard techniques. Results usingrecombinantly expressed rabies virus G glycoprotein showed that humanmonoclonal antibody 17C7 mapped to an epitope within the NH₃ terminal19-422 AA of the ectodomain of the rabies G glycoprotein. Humanmonoclonal clones 5G5, 2B10, 1E5, did not react in immunoblots withsoluble G glycoprotein fragments.

To further test the interaction of HuMab 17C7 with rabies glycoproteinsin vitro, the rabies virus glycoproteins from a variety of rabies virusstrains and isolates were cloned and expressed. Wild type CVS-11glycoprotein was initially cloned and expressed from the pcDNA3.1Myc/His (Invitrogen) mammalian expression vector and but at low levelsin transfected human cells. To overcome this low level of expression, acodon-optimized version of the ERA rabies glycoprotein-encoding gene(era-co) was engineered using art recognized techniques. Other Gproteins were also cloned (ERA (era-n), a Skunk isolate from California,USA (skunk-ca), and the bat isolates l borealis-Tennessee and l.cinereus-Arizona). Codon-optimization of the ERA glycoprotein-encodinggene led to a marked increase in the expression level as compared towild type ERA glycoprotein (FIG. 5A), and served as a useful reagent formany subsequent experiments.

HuMab 17C7 was determined to immunoprecipitate the glycoproteins fromsolubilized cells transfected with era-co (not shown), era-n, skunk-ca,l borealis-Tennessee and l. cinereus-Arizona isolates (FIG. 5B). Usingflow cytometry it was further shown that HuMab 17C7 also bound dosedependently to cells expressing the ERA-CO, ERA-N, L. borealis-Tennesseeand L. cinereus-Arizona glycoproteins on their cell surface (FIGS. 5Cand D). These data show that HuMab 17C7 binds specifically to rabiesvirus glycoproteins from multiple strains and isolates.

To better characterize the epitope that HuMab 17C7 recognizes, 17C7 wastested and determined to recognized a soluble version of the rabiesglycoprotein (amino acids 20-439) that did not possess the cytoplasmicor transmembrane domains of the glycoprotein. HuMAb 17C7 was alsodetermined to recognized a secreted, soluble form of the ERAglycoprotein (ERA-CO20-439) and the CVS-11 glycoprotein (CVS-1120-439)spanning amino acids 20-439 in ELISA (FIG. 6A). Surprisingly, HuMab 17C7recognized denatured ERA-CO20-439 and ERACO in an SDS-PAGE gel afterincubation in sample buffer containing reducing agents and SDS. However,the robustness of the signal was greatly enhanced when the samples wereprepared without the addition of reducing agents (FIG. 6B).

This recognition in SDS-PAGE was not observed for CVS-1120-439glycoprotein without reducing agents (FIG. 6C). These data indicate thatHuMab 17C7 recognizes a discontinuous epitope on the ERA rabiesglycoprotein. HuMab 17C7 recognizes minor site a and antigenic site IIIof the rabies virus glycoprotein.

To better understand which regions on the rabies glycoprotein arerecognized by HuMab 17C7, rabies viruses capable of growing in thepresence of HuMab 17C7 were engineered. In order to create HuMab 17C7resistant viruses a CVS-11 strain and the Skunk-CA isolate were cellcultured adapted. HuMab 17C7 resistant viruses from the CVS-11 virusstocks were isolated. Analysis of the glycoprotein-encoding sequences ofthese CVS-11 derived viruses revealed 3-point mutations in the 8 virusesanalyzed (CVS1 through 8). Interestingly, in two cases amino acidchanges at Asparagine 336, were identified. One virus contained a Asn toLys change, and multiple viruses contained an Asn to Asp change. Two ofthe viruses contained an Asn to Asp change at 336, as well as a Gln toLys change at 426. Asparagine 336 is within a region previouslyidentified as part of antigenic site III (Table 11).

In order to address whether Asparagine 336 was a residue critical forHuMab 17C7 binding, an Asparagine 336 residue in the ERA-co construct tothose observed in the CVS-11-derived resistant viruses was mutated. TheERA glycoprotein, as described in FIGS. 5 and 6, is robustly recognizedby HuMab 17C7; and the ERA virus, which is highly similar inglycoprotein sequence to Skunk-CA is also potently neutralized by HuMab17C7 as compared to CVS-11. Therefore, in this set of experiments theAsp 336 residue was shown to be important for HuMab 17C7 neutralizationof the CVS-11 virus and also important for maintaining the HuMab 17C7epitope within the ERA glycoprotein. The mutated glycoproteins ERA-CON336K and ERA-CO N336D were expressed and assayed for recognition byHuMab 17C7. The mutant glycoproteins were recognized by HuMab 17C7 in anELISA, however HuMab 17C7 binding to the ERA-CO N336K glycoprotein wasgreatly reduced compared to wild type (FIG. 7A). The levels of wild typeand mutant glycoprotein captured in the ELISA assay were similar, asshown by comparable binding of a mouse anti-rabies glycoproteinmonoclonal antibody (FIG. 7A). The mutant glycoproteins were all theappropriate molecular weights, as shown by immunoprecipitation using theHis tag, followed by immunoblot analysis with a Myc tag antibody (FIG.7B). HuMab 17C7 immunoprecipitated the ERA-CO and ERA-CO N336Dglycoproteins more readily than the ERA-CO N336K glycoprotein (FIG. 7B),which is consistent with the diminished binding of ERA-CO N336K observedin the ELISA. In contrast to wild type ERA-CO, the mutant proteins werenot recognized in Western blot under non-reducing conditions (FIG. 7B).We also created ERA-CO N336D Q426K and ERA Q426K, and the ELISA andimmunoblot results were similar to those for ERA-CO N336D, and ERA-COQ426K respectively, revealing that the Q426K mutation did not affectHuMab 17C7 binding.

In order to address whether recognition by HuMab 17C7 correlated withneutralization activity, a rabies glycoprotein pseudotyped HIV-1pseudovirus (10, 20), using the ERA-CO glycoprotein was created. It wasobserved that these pseudovirus particles infected human cells (FIG.8A), and that HuMab 17C7 potently inhibited infection by wild typeERA-CO pseudovirus, showing significant inhibition down to 100 pM (FIG.8B). Unrelated non-rabies HuMabs were tested at 1000 nM and did notneutralize rabies pseudovirus. Interestingly, HuMab 17C7 also inhibitedinfection of the ERA-CO N336K and ERA-CO N336D pseudoviruses (FIG. 8C),consistent with the observation that HuMab 17C7 recognizes ERA-CO N336Kand ERA-CO N336D glycoproteins.

Similar to HuMab 17C7, hRIG also inhibited all of the rabiespseudoviruses in a dose-dependant manner (FIG. 8D). These datademonstrate that the mutations that render the CVS-11 virus immune toHuMab 17C7 neutralization diminish, but do not abrogate, HuMab 17C7recognition of the ERA glycoprotein and neutralization of ERApseudovirus.

In order to test whether other well characterized antigenic sites wererecognized by HuMab 17C7 a panel of mutant glycoproteins containingamino acid changes previously reported for mAb-resistant viruses alteredin residues affecting antigenic sites I, II, III and minor site a werecreated (Table 11). HuMab 17C7 readily immunoprecipitated all of themutant glycoproteins from cell lysates with the exception of the R333I,K342T, G343E glycoprotein, which was mutated in a portion of antigenicsite III (a.a. 333) and minor site a (a.a. 342 and 343). It was furthercharacterized that the determinant important for HuMab 17C7 binding bycreating a separate R333I site III mutant and K342T, G343E minor site amutant. The R333I site III mutant was recognized by HuMab 17C7 in ELISAand immunoblot, while the K342T, G343E minor a and the R333I, K342T,G343E site III/minor a mutants were less well recognized 19 (FIGS. 9Aand B). The K342T, G343E and the R333I, K342T, G343E mutants wererecognized by a commercial rabies monoclonal antibody (FIG. 9A), andwere the appropriate molecular weight (FIG. 9B), indicating that theglycoproteins were expressed at comparable levels. Therefore, it wasdetermined that the lack of HuMab 17C7 binding to the minor site amutants was due to mutations in amino acids 342 and 343 of theglycoprotein, demonstrating that these amino acids are important forHuMab 17C7 recognition of the rabies glycoprotein (Table 12).

In addition, the glycoprotein sequences of rabies virus isolates andnon-rabies lyssaviruses at amino acids 336, 342 and 343 were compared.The residues important for HuMab 17C7 are conserved between divergentstrains of rabies virus and Australian bat lyysavirus, but not otherlyssaviruses (Table 13). The glycoprotein sequences of 154 rabiesviruses were compared from human, bat and carnivore isolates from allover the world, including North and South America, China and India.Sequence comparison revealed that Asparagine 336 was 93% conserved,Lysine 342 was 98% conserved and Glycine 343 was 99% conserved. Thesedata indicate that residues important for HuMab 17C7 recognition ofrabies virus glycoprotein are highly conserved.

TABLES 11 HuMab 17C7 Resistant Viruses Amino acid Amino Acid Proximityto Virus number change Codon Change antigenic site CVS1 336 Asn to LysAAT to AAG III CVS2-6 336 Asn to Asp AAT to GAT III CVS7-8 336 Asn toAsp AAT to GAT III CVS7-8 426 Glu to Asp CAG to AAG N/A

TABLE 12 HuMab 17C7 Recognizes Site I And Site II Mutated GlycoproteinsMutations in recombinant glycoprotein HuMab 17C7 binding Antigenic sitesR333I + III K342T, G343E − Minor a R333I, K342T, G343E − Minor a and IIIK226E, L231P + I G34E + II G40V, S42P, M44I + II K198E + II

TABLE 13 Amino Acids 330-345 of Rabies Viruses and Other LyssavirusesVirus Genbank ID Amino acids 330-345 CVS-11 AF085333 KSVRTWNEIIPSKGCLERA-CO AF406693 KSVRTWNEILPSKGCL Skunk-CA N/A KSVRTWNEILPSKGCLL. borealis-TN N/A KSVKTWNEVIPSKGCL L. cinereus-AZ N/A KSVKTWNEVIPSKGCLERA native N/A KSVRTWNEIIPSKGCL ABLV AF406693 KSVRTWNEIIPSKGCL EBLV-1AF298143 KSVREWTEVIPSKGCL EBLV-2 AF298145 KSIREWTDVIPSKGCL LagosAF429312 LKVDNWSEILPSKGCL Mokola MVU17064 KRVDRWADILPSRGCL

HuMab 17C7 recognizes a discontinuous epitope due to its ability to bindwith greater reactivity with non-reduced protein. The interaction ofHuMab 17C7 with the rabies glycoprotein is also unique because it isable to immunoprecipitate membrane bound glycoproteins of a variety ofrabies isolates, and to neutralize all of these isolates, but is onlyable to interact with a subset of secreted soluble glycoproteins inELISA and immunoblots.

The recognition of non-reduced protein by HuMab 17C7 indicates thatantigenic site II, minor site a, or an unknown conformationaldeterminant of the rabies glycoprotein is important for recognition byHuMab 17C7. The analysis of mutant glycoproteins revealed that 2 aminoacid changes at minor site a dramatically decreased HuMab 17C7recognition of the rabies glycoprotein. These two amino acid changesdisrupted the HuMab 17C7-binding site on the rabies glycoprotein and/orresult in a modification of the rabies glycoprotein tertiary structurecritical for HuMab 17C7 binding.

These data show that HuMab 17C7 resistant viruses indicate thatAsparagine 336 is important for HuMab 17C7 neutralization. The aminoacid change at residue 336 disrupts the HuMab 17C7-binding site on therabies glycoprotein and/or results in a modification of the rabiesglycoprotein structure critical for HuMab 17C7 binding.

The analysis of mutant glycoproteins created with site-directedmutagenesis also revealed that HuMab 17C7 recognizes both minor site aand part of antigenic site III.

Taken together these results indicate that HuMab 17C7 recognizes anepitope that is broadly conserved. The broad cross reactivity of HuMab17C7 indicates that it can be used in place of RIG for post exposureprophylaxis.

Example 5 Protection of Hamsters from Lethal Rabies Virus Challenge byAdministration of Anti-Rabies Virus Antibodies

Antibodies were tested for the ability to protect hamsters fromchallenge with a lethal dose of rabies virus (see Tables 14-15).

The human monoclonal antibody 17C7 was also tested in a hamster model ofpost exposure prophylaxis (PEP) to determine its potential as aprophylaxis for rabies virus infection in humans. Hamsters werechallenged in the gastrocnemius muscle of the hind leg with a fatal doseof rabies virus. The challenge virus was originally isolated from aTexas coyote. In this model, untreated animals die of rabies virusinfection in less than two weeks.

Briefly, animals were challenged in the gastrocnemius muscle with 50 μlof rabies virus and given anti-rabies virus antibodies in the same site24 hours later. Animals (n=9) were treated with a single dose of 19mg/kg of commercially available human rabies serum derivedimmunoglobulin (HRIG, Imogam, Aventis) or human monoclonal antibody 17C7at various doses (5, 0.5 or 0.25 mg/kg). All animals in an untreatedchallenge group died of rabies within 2 weeks of challenge. The percentsurvival at 63 days after challenge showed better protection by themonoclonal antibody at a dose of 0.25 mg/kg than commercially availablehuman immunoglobulin (Table 14).

A similar experiment was conducted where animals were treated withantibody post exposure to rabies and, in addition, treated with rabiesvaccine. Commercial human vaccine was administered in the oppositegastrocnemius muscle from the challenge site in a 50 μl injection volume1, 3, 7, 14 and 28 days after rabies challenge. Antibodies wereadministered as described previously. Again, the percent survival at 53days after challenge showed better protection by the monoclonal antibodyat a dose of 0.125 mg/kg than commercially available humanimmunoglobulin (Table 15).

Antibody was administered alone and with vaccine and results shown inTables 14-15 demonstrate that hamsters challenged with a lethal dose ofrabies virus can be protected with antibodies of the invention givenafter exposure to the virus either alone (see Table 14) or inconjunction with the administration of a rabies vaccine (Table 15).

To demonstrate that 17C7 does not interfere with vaccine response,hamsters were given 17C7 and rabies vaccine. As shown in Table 16, theanimals responded to vaccine even when given 17C7, thereby demonstratingthat the 17C7 antibody does not interfere with vaccine response.

TABLE 14 Post exposure protection from rabies with a human monoclonalantibody^(a) Sample IU/kg mg/kg Survivorship A human rabies immuneglobulin 15 8.0 5/9 B human rabies immune globulin 6 4.0 4/9 C humanrabies immune globulin 1 0.4 0/9 D human rabies immune globulin 0.05 0.00/9 E hu MoAb 17C7 26 1.7 9/9 F hu MoAb 17C7 7 0.9 9/9 G hu MoAb 17C7 10.1 6/9 H hu MoAb 17C7 0.05 0.0 1/9 I Controls — — 0/9 ^(a)At 24 hrsafter inoculation of a Texas coyote rabies virus isolate (#323),prophylaxis was initiated in eight treatment groups of 9 animals eachwith human monoclonal antibody 17C7 (26 IU/kg; 7 IU/kg, 1 IU/kg or 0.05IU/kg) or commercial human rabies immune globulin (15 IU/kg, 6 IU/kg, 1IU/kg or 0.05 IU/kg), administered at the site of virus inoculation. Theuntreated control group consisted of 9 animals.

TABLE 15 Rabies post-exposure prophylaxis including vaccine: comparisonof a human monoclonal antibody to human rabies immune globulin^(a)Sample IU/kg mg/kg Survivorship at 90 d A human rabies immune globulin20 21 17/18 B human monoclonal antibody 20 1 17/18 C human monoclonalantibody 10 0.5 16/18 D human monoclonal antibody 2 0.1 16/18 E controls— —  0/18 ^(a)At 24 hrs after rabies virus inoculation (50 ul of a1:1000 (10^(6.8) MICLD₅₀/ml) salivary gland homogenate from a naturallyinfected coyote (Texas coyote rabies virus isolate #323)), prophylaxiswas initiated in four treatment groups (A-D) of 18 hamsters each withhuman monoclonal antibody 17C7 (20 IU/kg; 10 IU/kg or 2 IU/kg) orcommercial human rabies immune globulin (20 IU/kg), administered at thesite of virus inoculation. A 50 ul volume of commercial rabies vaccinewas administered in the left gastrocnemius muscle. Additional doses ofvaccine were administered on days 3, 7, 14 and 28. The untreated controlgroup consisted of 18 animals.

TABLE 16 Geometric Mean liters of Rabies Virus Neutralizing AntibodiesFollowing Rabies Vaccine and Antibody Combinations Day Groups 3 7 14 2842 human rabies Ig + 15 26 1,315   10,013  5,878 vaccine +/−St Dev 9-2613-50 1241-1393 3730-26873 4293-8049 B hu MoAb + 12 22 339 4,442 6,704vaccine +/−St Dev 8-17 11-45  129-8889 2754-7158  6257-7189 C hu MoAb +10 17 404 9,304 5,812 vaccine +/−St Dev 9-11  9-31 181-900 1186-213784708-7161 ^(a)Three treatment groups (A-C) of animals received humanmonoclonal antibody 17C7 (25 IU/kg (group B) or 15 IU/kg (group C)) orcommercial human rabies immune globulin (25 IU/kg) administeredintramuscularly in the left gastrocnemius muscle. A 50 ul volume ofcommercial rabies vaccine was administered in the right gastrocnemiusmuscle. Additional doses of vaccine were administered on days 3, 7, 14and 28. On days 3, 7, 14, 28, and 42, six animals per group weresedated, blood was collected, and the animals were euthanized.

Taken together, these data indicate that HuMab 17C7 consistentlyprovides in vivo protection against rabies and can be used in place ofRIG for post exposure prophylaxis.

Example 6 Production of Anti-Rabies Virus Antibodies for Administrationin Humans

Human antibodies of the present invention can be cloned andrecombinantly expressed to facilitate or increase their production usingknown techniques.

Nucleic acid sequences encoding the variable heavy chain and lightchains of an antibody clone of the invention can be cloned into apIE-Ugamma1F vector using standard recombinant DNA methodology. Thevector is amplified in E. coli, purified, and transfected into CHOcells. Transfected cells are plated at 4×10⁵ cells per well in a 96-welldish and selected for vector transfection with G418. Resistant clonesselected by G418 resistance, are then assayed along with othertransfectomas for production of IgG. The expression of an antibody canbe amplified by growth in the presence of increasing concentrations ofmethotrexate. A culture capable of growth in 175 nM methotrexate ischosen for cloning single cells for further development. Plating theculture in 96 well plates at low density allowed generation of culturesarising from a single cell or clones. The cultures are screened forproduction of human IgG, and the cell that produces the highest level ofIgG is typically selected for further use. The methotrexate-amplifiedclone is expanded to produce a cell bank including multiple frozen vialsof cells. Alternatively, glutamine synthetase (GS) vectors can be usedwith cell selection achieved using, e.g., methionine sulphoximine (see,e.g., U.S. Pat. Nos. 5,827,739; 5,122,464; 5,879,936; and 5,891,693).

To prepare antibodies from transfected cells, cells from a cloneisolated in the previous steps are cultured and expanded as inoculum fora bioreactor. The bioreactor typically holds a 500 liter volume ofculture medium. The cells are cultured in the bioreactor until cellviability drops, which indicates a maximal antibody concentration hasbeen produced in the culture. The cells are removed by filtration. Thefiltrate is applied to a protein A column. Antibodies bind to thecolumn, and are eluted with a low pH wash. Next, the antibodies areapplied to a Q-Sepharose column to remove residual contaminants, such asCHO cell proteins, DNA, and other contaminants (e.g., viralcontaminants, if present). Antibodies are eluted from the Q-Sepharosecolumn, nano-filtered, concentrated, and washed in a buffer such as PBS.The preparation is then aseptically aliquoted into vials foradministration.

Other Embodiments

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated monoclonal antibody that binds to rabies virus G protein,wherein the antibody comprises a variable heavy chain region comprisingresidues 20-144 of SEQ ID NO:1 and a variable light chain regioncomprising residues 21-127 of SEQ ID NO:2.
 2. The isolated monoclonalantibody of claim 1, wherein the antibody comprises a human, humanizedor chimeric antibody.
 3. The isolated monoclonal antibody of claim 1,wherein the antibody comprises a full length antibody.
 4. The isolatedmonoclonal antibody of claim 1, wherein the antibody is selected fromthe group consisting of a Fab, F(ab′)2, FV or a single chain Fvfragment.
 5. A composition comprising the antibody of claim 1 in apharmaceutically acceptable carrier.
 6. The composition of claim 5,further comprising one or more additional antibodies.
 7. A method oftreating rabies virus disease in a subject, the method comprising:administering to the subject the antibody of claim 1 in an amounteffective to inhibit a symptom of rabies virus disease.
 8. The method ofclaim 7, wherein the antibody is administered in combination with one ormore additional antibodies.
 9. A method of producing the antibody ofclaim 1, comprising transfecting an isolated host cell with one or morevectors encoding the heavy and light chains of the antibody andisolating the expressed antibody.
 10. An isolated monoclonal antibodythat binds to rabies virus G protein, wherein the antibody comprises aheavy chain region comprising residues 20-144 of SEQ ID NO:1.
 11. Theisolated monoclonal antibody of claim 10, wherein the antibody comprisesa human, humanized or chimeric antibody.
 12. The isolated monoclonalantibody of claim 10, wherein the antibody comprises a full lengthantibody.
 13. The isolated monoclonal antibody of claim 10, wherein theantibody is selected from the group consisting of a Fab, F(ab′)2, FV ora single chain Fv fragment.
 14. A composition comprising the antibody ofclaim 10 in a pharmaceutically acceptable carrier.
 15. The compositionof claim 14, further comprising one or more additional antibodies.
 16. Amethod of producing the antibody of claim 10, comprising transfecting anisolated host cell with a vector encoding the heavy chain of theantibody and isolating the expressed antibody.
 17. An isolatedmonoclonal antibody that binds to rabies virus G protein, wherein theantibody comprises a light chain region comprising residues 21-127 ofSEQ ID NO:2.
 18. The isolated monoclonal antibody of claim 17, whereinthe antibody comprises a human, humanized or chimeric antibody.
 19. Theisolated monoclonal antibody of claim 17, wherein the antibody comprisesa full length antibody.
 20. The isolated monoclonal antibody of claim17, wherein the antibody is selected from the group consisting of a Fab,F(ab′)2, FV or a single chain Fv fragment.
 21. A composition comprisingthe antibody of claim 17 in a pharmaceutically acceptable carrier. 22.The composition of claim 21, further comprising one or more additionalantibodies.
 23. A method of producing the antibody of claim 17,comprising transfecting an isolated host cell with a vector encoding thelight chain of the antibody and isolating the expressed antibody.